Propofol Pretreatment Attenuates Aquaporin-4 Over-Expression and Alleviates Cerebral Edema After

Propofol Pretreatment Attenuates Aquaporin-4
Over-Expression and Alleviates Cerebral Edema After
Transient Focal Brain Ischemia Reperfusion in Rats
Yue-Ying Zheng, MS*
Yun-Ping Lan, MS*
Hui-Fang Tang, PhD†
Sheng-Mei Zhu, MD, PhD*
BACKGROUND: Cerebral edema is a major threat for stroke victims. Most studies have
focused on the neuroprotective activities of propofol, addressing infarct volume
rather than cerebral edema. Aquaporin-4 (AQP4) plays an important role in
maintaining brain water homeostasis under various neurological insults. We
explored the effect of propofol pretreatment on cerebral edema in a rat model of
brain ischemia reperfusion and assessed the involvement of AQP4.
METHODS: To induce brain ischemia reperfusion, we introduced a silicone-coated
monofilament nylon suture into the origin of the middle cerebral artery, withdrawing it after 90 min. Treatment groups (n ⫽ 32), received propofol (0.1
mL 䡠 kg⫺1 䡠 min⫺1) infusion for 30 min before occlusion; the vehicle group (n ⫽ 32)
and the sham-operated group (n ⫽ 28), which received the intralipid vehicle at the
same time and rate. To assess cerebral infarct volume, we used 2, 3, 5-triphenyltetrazolium chloride staining; wet– dry weight ratio was the basis for cerebral
edema estimation, and we used immunohistochemistry and Western blot to detect
AQP4 expression.
RESULTS: The wet– dry weight ratio decreased from 86.89% ⫾ 0.71% in the vehicle
group (n ⫽ 6) to 72.42% ⫾ 0.74% in the propofol group (n ⫽ 6), corresponding to
an average decrease of 16%. In parallel and based on immunohistochemical
semi-quantification, the propofol group exhibited remarkable attenuation of AQP4
over-expression in the ischemic border zone compared with the vehicle group:
1.28 ⫾ 0.03 vs 1.40 ⫾ 0.05, n ⫽ 7, respectively; P ⬍ 0.05. Values derived from
Western blot quantification were similarly decreased in the propofol group
compared to the vehicle group: 20.85% ⫾ 4.18% vs 31.67% ⫾ 3.23%, n ⫽ 4,
respectively; P ⬍ 0.05. However, infarct volume and neurologic deficit in postischemic rats in the propofol group were not statistically different from values in the
vehicle group.
CONCLUSIONS: We conclude that prestroke treatment with propofol reduces postischemic cerebral edema in rats, possibly through inhibiting AQP4 over-expression
in the boundary zone of ischemia.
(Anesth Analg 2008;107:2009 –16)
ropofol (2, 6-dilsopropylphenol) is an IV anesthetic extensively used in clinical practice and characterized by its rapid induction and quick patient recovery
from its effects. In addition, propofol is in widespread use
as a sedative for intensive care patients. Results of in vitro
From the *Department of Anesthesiology, the First Affiliated
Hospital, School of Medicine, Zhejiang University, People’s Republic of China; and †Department of Pharmacology, School of Medicine, Zhejiang University, Hang Zhou, People’s Republic of China.
Accepted for publication July 18, 2008.
Supported by the Zhejiang Provincial Science Technology Foundation of China (No.2006C33066) and the Medical Science Research
Foundation of Health Bureau of Zhejiang Province of China (No.
Address correspondence and reprint requests to Sheng-Mei Zhu,
MD, PhD, Department of Anesthesiology, The First Affiliated
Hospital, School of Medicine, Zhejiang University, 79 Qingchun
Road, 310003, HangZhou, People’s Republic of China. Address
e-mail to [email protected].
Copyright © 2008 International Anesthesia Research Society
DOI: 10.1213/ane.0b013e318187c313
Vol. 107, No. 6, December 2008
and in vivo studies are inconsistent regarding the neuroprotective activities of propofol and possible mechanisms of
action; suggestions have included reduction in cerebral
metabolism,1 antioxidant activity,2 antiexcitotoxic properties,3 modulation of inhibitory neurotransmitter action and
excitatory neurotransmitters,4,5 or influence on neuronal
apoptosis.6,7 Therefore, the impact of propofol on cerebral
ischemia and its mechanism of action remain to be clarified.
Cerebral edema is a serious complication in association with stroke.8,9 The sequelae are severe, with
increased intracranial pressure and herniation not
only aggravating neurologic outcome but also threatening life.10 Results of studies on the antiedema effect
of propofol are less frequently reported. Some surgical
procedures, such as carotid endarterectomy and aneurysmal clipping, carry an enhanced risk of cerebral
edema; in addition, some high-risk patients with preexisting cerebrovascular disease are prone to cerebrovascular occlusion events in the perioperative period.
Thus, if one of the anesthetic outcomes of propofol
Figure 1. The whole experimental procedure
and the flow chart. Arterial blood pressure
measurement and blood sampling were performed 30 min before the onset of ischemia, 15
min after propofol administration, 15 min after
ischemia and 15 min after reperfusion (A). (B)
shows that 92 animals were randomized to 3
groups and further subdivided for various
administration includes alleviation of edema induced
by postoperative stroke, these findings would be
Aquaporin-4 (AQP4), a member of the transmembrane water channel protein family, is involved in
cerebral edema and brain water homeostasis. AQP4
is strongly enriched at the brain– blood and brain–
cerebral spinal fluid interfaces.11,12 Changes in AQP4
in animals coincide with cerebral edema development
evoked by brain ischemic reperfusion injury.13,14
AQP4 deficiency reduces cytotoxic edema resulting
from focal brain ischemia or water intoxication15,16 but
aggravates vasogenic cerebral edema induced by
brain tumor, freeze injury, or intraparenchymal fluid
In this study, we explored whether propofol pretreatment reduces cerebral edema in a rat model of
brain ischemia reperfusion and the involvement of
The Medical Faculty Ethics Committee of Zhejiang
University approved the study protocol. All experimental procedures were performed in accordance
with the Guide for the Care and Use of Experimental
Animals. Male Sprague-Dawley rats (230 –280 g),
obtained from the Experimental Animal Center in
the Zhejiang Academy of Medical Sciences, were
allowed free access to water and laboratory chow
before experimentation.
The experimental procedure and a flow chart are
showed in Figure 1. Rats were anesthetized with
chloral hydrate (400 mg/kg, ip) and then allowed to
breathe spontaneously in oxygen-enriched air (fraction of inspired oxygen [Fio2]: 40%) through a facemask. The left femoral artery was cannulated with a
polyethylene tube for arterial blood pressure monitoring (PC-Lab, Kelong, Nanjing, China) and blood sampling, and the left femoral vein was used for drug
administration. Arterial blood pH, Pao2, and Paco2
were monitored by Blood Gas Analyzer (Blood Gas
Analyzer ABL 330, Leidu, Denmark) and blood glucose with a OneTouch Basic Blood Glucose Monitoring System (LifeScan). The physiological variables
were recorded before the operation (as baseline), 15
min after drug exposure, 15 min after ischemia, and 15
min after reperfusion. Rectal temperature was maintained at 37.0 ⫾ 0.5°C with a heating pad throughout
the surgical procedures.
Rats were then randomly assigned to one of three
groups. The propofol group (n ⫽ 32) received
propofol (2,6-diisopropylphenol) in an intralipid
emulsion (Diprivan®, Astra-Zeneca, Caponago, Milano, Italy, 10 mg/mL) IV infused at a rate of 0.1
mL 䡠 kg⫺1 䡠 min⫺1 for 30 min until the onset of the
middle cerebral artery occlusion (MCAo). The vehicle group (n ⫽ 32) and the sham-operated group
(n ⫽ 28) received the lipid vehicle only (Intralipid,
Fresenius Kabi, Verona, Italy) at the same dose and
Propofol Lessens AQP-4 Over-Expression and Cerebral Edema
We then prepared the animals for MCAo as previously described.18 Briefly, after a midline cervical
incision, the right external carotid artery as well as its
branches were carefully exposed, ligated, and cut. A
0.2-mm silicone-coated monofilament nylon suture
(diameter of the round tip: 0.26 – 0.28 mm) was introduced into the right internal carotid artery through the
right external carotid artery stump. We used laser
Doppler flowmetry (ML191, AD Instruments Pty) to
measure regional cerebral blood flow, as reported.19 A
sudden decrease of regional cerebral blood flow from
100% (before ischemia) to 50% or less confirmed
correct placement of the suture. Regional cerebral
blood flow returned to 98% ⫾ 8% with removal of the
suture 90 min after occlusion to allow reperfusion.
Finally, after ligation of the right external carotid
artery and closure of the skin incision, the animal
recovered. After awakening from anesthesia, the animals were maintained in an air-conditioned room at
24°C. Except for the lower depth of the inserted
intraluminal suture, the sham-operated animals underwent the same surgical process.
At 24 h of reperfusion, a single observer blinded to
group assignment put the rats through a neurologic
examination, grading each animal on a scale of 0 – 4, as
previously reported.18 The criteria were as follows:
0 ⫽ no observable deficit; 1 ⫽ left forelimb flexion or
failure to extend fully when lifting the animal by the
tail; 2 ⫽ circling to the left side but normal posture at
rest; 3 ⫽ leaning to the left side at rest; 4 ⫽ unable to
walk spontaneously or stroke-related death. Animals
included in the subsequent study had a neurologic
score ⬎1 for the rats subjected to the MCAo or ⫽ 0 for
the sham-operated rats. These rats were anesthetized
with chloral hydrate and decapitated. After rapid
removal, the brains (n ⫽ 8 for the propofol group and
the vehicle group, respectively; n ⫽ 6 for the shamoperated group) were sectioned coronally into slices 2
mm thick by a special brain matrix, then stained with
0.05% 2,3,5-triphenyl-tetrazolium chloride at 37°C for
30 min in the dark.20 After fixation of the slices in 0.1
M phosphate-buffered 10% formaldehyde for 24 h at
room temperature, we used a digital camera to obtain
images of the brain slices, which we traced and
quantified using Image Tool 3.0. To reduce the impact
of brain edema on infarct area, we subtracted the
noninfarct area in the right hemisphere from the total
area of the left hemisphere to obtain the infarct area in
the right ischemic hemisphere in each slice.21 To
calculate the total infarct volume in each brain, we
multiplied the thickness (2 mm) for the infarct area of
each brain slice in the same brain and then summed
these values. Values used for analysis were thus the
absolute total infarct volume (mm3) and the percentage of infarct brain volume over total brain volume.
To quantify cerebral edema, we used the wet– dry
weight method. After rapid removal 24 h after
ischemia–reperfusion injury, the brains (n ⫽ 6 rats for
each group) were divided into the ipsilateral and
Vol. 107, No. 6, December 2008
contralateral hemispheres. Each brain tissue was
placed on a piece of aluminum foil. After the wet
weight of the hemisphere was measured, the tissue
was dried at 110°C for 24 h to obtain the dry weight.
We calculated the brain water content in the ischemic
hemisphere using the following equation: %water ⫽
(wet weight ⫺ dry weight)/wet weight ⫻ 100%.22
In addition, 8-␮m coronal sections (beginning at the
bregma and extending 4 mm caudally) were cut from
each brain from the included rats (n ⫽ 8 for the
propofol group and the vehicle group, respectively;
n ⫽ 6 for the sham-operated group) with a cryomicrotome (CM3050, Leica, Germany) for immunohistochemical examination. After incubation with 0.3%
hydrogen peroxide for 10 min, sections were blocked
with 2% normal goat serum for 10 min at room
temperature. Then brain sections were placed overnight at 4°C with anti-AQP4 (Chemicon; rabbit, 1/200)
followed by polyperoxidase-anti-mouse/rabbit IgG
(PV-900 polymer detection system, Zhongshan Biotechnology CO, China) for 1 h at 37°C. After each
incubation, sections were rinsed with phosphate buffered saline for 3 ⫻ 5 min. Finally, diaminobenzidine
was added to the sections. Phosphate buffered saline
took the place of the primary antibody as the negative
We used an optical microscope to photograph the
diaminobenzidine staining as an indicator of immunolabeling. To semi-quantify AQP4 expression, we
used optical density (OD) measurements in three
different fields in both the border region of ischemia
and the corresponding part of the contralateral hemisphere (⫻200). A pathological image analysis system
allowed detection of the OD (Hpias-1000, Wuhan,
China), which we used to calculate a ratio of OD
between the ischemia hemisphere and the contralateral hemisphere, as reported.13 An independent observer from the pathology department performed the
To quantify AQP4 expression further, we performed Western blot analysis. At 24 h of reperfusion,
cortical brain tissues from the boundary zone adjacent
to the ischemic core in both the propofol and vehicle
groups were dissected on ice as described23 and stored
at ⫺70°C until use; the corresponding tissue in the
sham-operated group was also dissected (n ⫽ 4 rats
for each group). Tissues were homogenized using a
Polytron homogenizer in RIPA buffer (Kangchen Biotechnology, Shanghai, China) containing protease inhibitor cocktail (P8340, Sigma). After homogenization
and centrifugation at 12000g at 4°C for 30 min, the
resultant supernatant was collected. Protein samples
(50 ␮g) were subjected to 12% Sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred to
nitrocellulose membranes. The membranes were
blocked by 5% fat-free milk, then incubated with a
rabbit polyclonal antibody against AQP4 (Chemicon;
1/500) or a rabbit monoclonal antibody against ␤-actin
(Sigma; 1/1000) in Tris-Buffered Saline Tween-20
© 2008 International Anesthesia Research Society
Table 1. Summary of Physiological Parameters at Sampling Time Points
MAP (mm Hg)
15 min after
15 min after
15 min after
Pao2 (mm Hg)
15 min after
15 min after
15 min after
Paco2 (mm Hg)
15 min after
15 min after
15 min after
Glucose (g/L)
15 min after
15 min after
15 min after
15 min after
15 min after
15 min after
Sham group (n ⫽ 6)
Vehicle group (n ⫽ 6)
Propofol group (n ⫽ 5)
drug exposure
123 ⫾ 3
119 ⫾ 3
116 ⫾ 6
117 ⫾ 4
119 ⫾ 8
121 ⫾ 5
123 ⫾ 2
118 ⫾ 9
118 ⫾ 7
109 ⫾ 4
115 ⫾ 9
128 ⫾ 7
drug exposure
146 ⫾ 6
137 ⫾ 3
135 ⫾ 2
142 ⫾ 5
139 ⫾ 2
141 ⫾ 3
133 ⫾ 2
136 ⫾ 3
140 ⫾ 4
135 ⫾ 3
136 ⫾ 5
144 ⫾ 7
drug exposure
43.2 ⫾ 1.7
41.0 ⫾ 1.5
42.7 ⫾ 1.9
43.5 ⫾ 1.6
44.8 ⫾ 1.5
40.6 ⫾ 1.3
40.0 ⫾ 1.6
42.9 ⫾ 1.4
44.7 ⫾ 1.8
42.2 ⫾ 1.5
44.0 ⫾ 1.6
40.1 ⫾ 2.0
drug exposure
6.05 ⫾ 0.98
6.24 ⫾ 0.98
6.12 ⫾ 0.62
6.19 ⫾ 0.74
5.65 ⫾ 0.30
5.89 ⫾ 0.44
6.15 ⫾ 0.39
5.99 ⫾ 0.40
5.95 ⫾ 0.40
6.23 ⫾ 0.27
6.32 ⫾ 0.19
5.92 ⫾ 0.40
drug exposure
7.38 ⫾ 0.02
7.36 ⫾ 0.02
7.34 ⫾ 0.03
7.35 ⫾ 0.01
7.34 ⫾ 0.03
7.40 ⫾ 0.03
7.36 ⫾ 0.02
7.38 ⫾ 0.01
7.40 ⫾ 0.04
7.38 ⫾ 0.03
7.36 ⫾ 0.02
7.35 ⫾ 0.01
Values are expressed as mean ⫾ SEM.
Repeated-measures ANOVA with the Least.
Significant Difference Post Hoc test was used.
MAP ⫽ mean arterial blood pressure; PaO2 and PaCO2 the pressure of O2 and CO2 in arterial blood.
overnight at 4°C. After washing, the membranes were
reacted with the horseradish peroxidase-conjugated
antirabbit IgG (1:1000, Zhongshan, China) for 2 h at
room temperature. Immunoreactive bands were visualized by ECL chemiluminescence (ECL Kit, Santa
Cruz Biotechnology) and exposed on radiograph film
to reveal the AQP4 band (30 kDa) and ␤-actin (43
kDa). The integrated densities value was analyzed
with a computerized image analysis system (Gel-pro
Analyzer 4.0, Media Cybernetics). The results of AQP4
expression were expressed as the percentage change
over ␤-actin.
The sample size calculation was based on previous
studies. The primary end-point of the study was
wet– dry weight ratio. Paczynski et al.,24 in a rat model
of ischemic stroke, showed that the mean and standard deviation of wet-dry weight ratio was 83.3% and
0.85%, respectively. We assumed that propofol can
reduce cerebral edema assessed by wet-dry weight
ratio by 2%. Based on these data, a sample size of 5
rats was needed for each group to detect a difference
of 2% among the 3 groups with a type I error of 0.05
and a power of 0.85, using Turkey HSD test. Allowing
for possible dropouts, we chose to use six rats for each
group. We arbitrarily chose a similar sample size for
the other subgroups.
Statistical analysis was performed among propofol,
vehicle and sham-operated groups and their respective subgroups with the statistical software (SPSS 13.0
for Windows, SPSS). Data are presented as Mean ⫾
sem. We used one-way analysis of variance (ANOVA)
followed with the Least Significant Difference Post Hoc
test for most parameters, except that we compared the
neurological deficit scores with the nonparametric
Mann-Whitney U-test, the physiological parameters
with repeated measures ANOVA with the Least Significant Difference Post Hoc test, and mortality using
the ␹2 test. A P value ⬍0.05 was considered statistically significant.
Table 1 shows the physiologic variables. Propofol
pretreatment decreased mean arterial blood pressure
15 min after propofol infusion, but the change was not
statistically significant. Arterial blood gas variables
and blood glucose concentrations among the three
groups at all sampling points also did not differ
Of the original 92 animals, 3 died before the following examination: 1 in the vehicle group (3% mortality)
and 2 in the propofol group (6% mortality). In addition, we excluded one rat each from the propofol
group and the vehicle group because of failure to meet
the neurologic deficit score criteria.
The neurologic evaluation of rats at 24 h after
transient MCAo reperfusion revealed that, compared
with the vehicle (n ⫽ 6), propofol pretreatment (n ⫽ 6)
tended to improve the neurologic outcome, but the
difference did not rise to the level of statistical significance (P ⫽ 0.165, Mann-Whitney U-test).
Propofol Lessens AQP-4 Over-Expression and Cerebral Edema
Figure 2. Effects of propofol pretreatment on edema formation induced by ischemia reperfusion injury in rats. Brain
edema was determined by the increase of ischemic hemisphere water content (n ⫽ 6 for each group). Results are
expressed as mean ⫾ sem *P ⬍ 0.05 versus sham operation
group; **P ⬍ 0.01 versus sham operation group; #P ⬍ 0.05 vs
vehicle group; ##P ⬍ 0.01 versus vehicle group; one-way
We found no statistical differences between the
vehicle and propofol groups in absolute infarct volume (278.97 ⫾ 36.25 mm3, n ⫽ 7 vs 223.3 ⫾ 67.37 mm3,
n ⫽ 7, one-way ANOVA, P ⫽ 0.351) or in the percentage of nonviable tissue volume over the total brain
volume (17.05% ⫾ 1.20% vs 13.77% ⫾ 3.87%, P ⫽
0.241), which we calculated by measuring consecutive
coronal slices stained by 2,3,5-triphenyl-tetrazolium
The ischemic hemisphere in the propofol group
exhibited a significantly lower water content increase
compared with vehicle: 72.42% ⫾ 0.74% vs 86.89% ⫾
0.71%, (P ⫽ 0.000; one-way ANOVA, n ⫽ 6 rats/group;
Fig. 2), corresponding to an average decrease of 16%.
In Figure 3B(a– c), representative low-magnification
pictures show expression of AQP4 mainly on astrocytic endfeet around brain vessels, with changes in
expression in the border zone of the lesion after brain
ischemia-reperfusion injury. The borders of the ischemia are represented in a schematic drawing (Fig. 3A).
The OD ratio in the vehicle group (n ⫽ 7) was
significantly higher than in the sham-operated group
(n ⫽ 6). However, the ratio in the propofol group (n ⫽
7) was significantly smaller compared to that of the
vehicle group (1.28 ⫾ 0.03 vs 1.40 ⫾ 0.05, P ⫽ 0.02,
one-way ANOVA, Fig. 3C).
Western blot analysis confirmed the immunohistochemistry results, showing the AQP4 protein as a
single 30-kDa band (Fig. 4A). Control blots without
primary antibody to AQP4 showed no specific AQP4
bands (data not shown). Compared with shamoperated rats, AQP4 protein concentration in the
boundary zone of the lesion increased significantly at
24 h after reperfusion in both the vehicle and propofol
groups. Propofol pretreatment markedly reduced
AQP4 over-expression compared with intralipid vehicle treatment (20.85% ⫾ 4.18% vs 31.67% ⫾ 3.23%,
P ⫽ 0.041, one-way ANOVA; n ⫽ 4 for each group;
Fig. 4B).
Vol. 107, No. 6, December 2008
Our objective was to characterize the effects of
propofol pretreatment prior to ischemia in inhibiting
formation of brain edema. We used MCAo as an
experimental model to induce brain edema and found
that MCAo-90 min followed by reperfusion for 24 h
elicited brain edema and upregulated AQP4 expression in the penumbra region. Propofol infusion before
ischemia ameliorated brain edema and attenuated
AQP4 over-expression. However, we did not observe
that propofol significantly improved neurologic deficits or decreased brain infarct volumes.
According to previous reports, 24-h postischemia is
the time of peak edema,25 leading us to choose this
time point for our study. AQP4 is a bidirectional water
channel protein, concentrated in cerebral spinal fluid–
brain and blood-brain interfaces. Our study showed
that MCAo-90 min and reperfusion at 24 h caused
AQP4 upregulation in rats, as detected by immunochemistry and Western blot analysis. However, others
have also demonstrated changes in AQP4 levels after
ischemia using other models. In a model of MCAo-30
min in mice, AQP4 over-expression in the periinfarct
zone was not significant at 24 h after reperfusion but
was at 48 h.13 AQP4 expression conversely decreased
23 h after brain transient ischemia 90 min.26 These
varying results may have been from differences in
species used (mouse versus rat), age of animals studied (young versus adult), or the severity of the ischemia and reperfusion (different duration in ischemia
or reperfusion). Meanwhile, we identified AQP4
down-regulation in a rat MCAo model in the propofol
group accompanied by an obvious reduction in cerebral edema. According to the classification of cerebral
edema based on the pathophysiologic mechanism,
cytotoxic edema develops by the time point of reperfusion at 24 h.27 Others have speculated that AQP4
over-expression enhances a rapid influx of water and
is responsible for the development of cytotoxic edema
after focal brain ischemia28; thus, AQP4 downregulation may alleviate cerebral edema.
No studies of which we are aware have clearly
connected the action of propofol and AQP4. The
mechanism may involve reversible protein phosphorylation and amino acid neurotransmitters. Protein
kinase C is one of the potential phosphorylation sites
of AQP4, and water permeability activity alters with
its phosphorylation.29 Protein kinase C activator inhibited ischemia-induced up-regulation of AQP4 and
attenuated brain swelling in a MCAo model 30 Moreover, protein kinase C–mediated protein phosphorylation is a target for general anesthetic effects in the
central nervous system, including propofol.31 Therefore, protein kinase C can reasonably be suggested as
a possible key location for propofol regulation of
AQP4 expression during brain ischemia-reperfusion
© 2008 International Anesthesia Research Society
Figure 3. Effects of propofol pretreatment on aquaporin-4 (AQP4) expression at 24 h after middle cerebral artery occlusion
(MCAo) reperfusion in rats. (A) The boxes in the schematic drawing represent the areas selected as the border of the lesion.
(B) Representative low-magnification images illustrate AQP4 immunohistochemical staining in the ischemic boundary zone
of an injured animal that received the intralipid vehicle (a, n ⫽ 7), an injured animal that received propofol (b, n ⫽ 7), and
a sham-operated animal that received intralipid (c, n ⫽ 6). “Phosphate buffered saline” is a negative control (d). (C) AQP4
expression was evaluated by calculating the ratio of the optical density of AQP4 immunolabeling in the ischemic hemisphere
(ISCH) and in the control hemisphere (CTL). The ratio for AQP4 in the propofol group significantly decreased compared with
the vehicle group. Results are mean ⫾ sem **P ⬍ 0.01 versus sham-operated group; #P ⬍ 0.05 versus vehicle group, one-way
Figure 4. Effects of propofol pretreatment on aquaporin-4
(AQP4) expression as revealed by Western blot (A, n ⫽ 4 for
each group); data are summarized in (B). The AQP4 band
migrated at 30 kDa, and AQP4 expression was downregulated with infusion of propofol at a rate of 0.1
mL 䡠 kg⫺1 䡠 min⫺1 prior to ischemia compared with intralipid administration (“vehicle”). Results are expressed as
mean ⫾ sem *P ⬍ 0.05 versus sham-operated group, #P ⬍
0.05 versus vehicle group, one-way ANOVA.
Another related factor may be the amino acid
neurotransmitter, glutamate. One study showed that
astrocytic swelling that glutamate elicited seemed to
correlate with AQP4.32 Knock-out AQP4 in mice
results in altered glutamate levels in the brain.33
Nevertheless, propofol could decrease extracelluar
glutamate levels by many possible mechanisms.34 –36
Glutamate might therefore be another factor in the
mechanism by which propofol regulates AQP4 expression. These ideas need to be further verified in
vitro and in vivo.
We did not identify a significant neuroprotective
role of propofol, a finding consistent with the results
of Ridenour et al.37 They demonstrated that propofol
had no greater neuroprotective effect than their halothane control, which was not recognized as a neuroprotectant. In contrast, other authors have reported
that propofol pretreatment reduced infarct area
and improved neurologic deficits after incomplete
ischemia-reperfusion injury.1,38 Again, differences in
species, study models, baseline anesthetics, and the
timing and dose of infused propofol may explain these
discrepancies. Further study is necessary to optimize
Propofol Lessens AQP-4 Over-Expression and Cerebral Edema
the propofol dose, the time and the sample size to
achieve maximum benefit. In addition, another possible explanation for the differences could be neurologic scoring. Although the neurologic scoring used
here has been extensively applied, it has never been
validated for it seems able to differentiate among
various infarct volumes.39 A six-point neuro-scoring
displayed a more significant correlation with infarct
volume40 and might be more suitable for such studies.
Meanwhile, a slightly later observation time might
improve evaluation of the neuroprotective potential of
We used rectal temperature monitoring in this
experiment because transcranial temperature measuring was not available. Pericranial temperature monitoring is more suitable than rectal temperature in
terms of experimental neuroprotection41; however,
because core temperature stayed within a narrow range
among the three groups, we infer that there would be no
difference in the brain-to-core-temperature relationship
among the groups. Because there is no absolute relationship between the electroencephalographic suppressive
effect and the neuroprotective effect of propofol,5 we did
not monitor electrical status.
In conclusion, regulation of AQP4 levels could be an
important mechanism in maintaining brain water homeostasis. Propofol pretreatment exerts an antiedema
effect in rats in a MCAo model, an effect that is partly
correlated with AQP4 down-regulation in the border of
the infarct zone. This study may open new avenues of
investigation into the antiedema effect of propofol.
We thank Dr. Wei-Zhong Gu for assisting in analyzing
immunohistological results and Yu Zhou for establishing the
MCAo model in the rat.
1. Kochs E, Hoffman WE, Werner C, Thomas C, Albrecht RF,
Schulte am Esch J. The effects of propofol on brain electrical
activity, neurologic outcome, and neuronal damage following
incomplete ischemia in rats. Anesthesiology 1992;76:245–52
2. Ergun R, Akdemir G, Sen S, Tasci A, Ergungor F. Neuroprotective effects of propofol following global cerebral ischemia in
rats. Neurosurg Rev 2002;25:95– 8
3. Hans P, Bonhomme V, Collette J, Albert A, Moonen G. Propofol
protects cultured rat hippocampal neurons against N-methylD-aspartate receptor-mediated glutamate toxicity. J Neurosurg
Anesthesiol 1994;6:249 –53
4. Ito H, Watanabe Y, Isshiki A, Uchino H. Neuroprotective
properties of propofol and midazolam, but not pentobarbital, on
neuronal damage induced by forebrain ischemia, based on the
GABAA receptors. Acta Anaesthesiol Scand 1999;43:153– 62
5. Wang J, Yang X, Camporesi CV, Yang Z, Bosco G, Chen C,
Camporesi EM. Propofol reduces infarct size and striatal dopamine accumulation following transient middle cerebral artery
occlusion: a microdialysis study. Eur J Pharmacol 2002;452:
303– 8
6. Englelhard K, Werner C, Eberspacher E, Pape M, Stegemann U,
Kellermann K, Hollweck R, Hutzler P, Kochs E. Influence of
propofol on neuronal damage and apoptotic factors after incomplete cerebral ischemia and reperfusion in rats: a long-term
observation. Anesthesiology 2004;101:912–7
Vol. 107, No. 6, December 2008
7. Iijima T, Mishima T, Akagawa K, Iwao Y. Neuroprotective effect
of propofol on necrosis and apoptosis following oxygen-glucose
deprivation–relationship between mitochondrial membrane potential and mode of death. Brain Res 2006;1099:25–32
8. Bounds JV, Wiebers DO, Whisnant JP, Okazaki H. Mechanisms
and timing of deaths from cerebral infarction. Stroke 1981;12:
474 –77
9. Steiner T, Ringleb P, Hacke W. Treatment options for large
hemispheric stroke. Neurology 2001;57(Suppl 1):S61—S68
10. Unterberg AW, Stover J, Kress B, Kiening KL. Edema and brain
trauma. Neuroscience 2004;129:1021–9
11. Rash JE, Yasumura T, Hudson CS, Aqre P, Nielsen S. Direct
immunogold labeling of aquaporin-4 in square arrays of astrocyte and ependymocyte plasma membranes in rat brain and
spinal cord. Proc Natl Acad Sci USA 1998;95:11981– 6
12. Verkman AS. Aquaporin water channels and endothelial cell
function. J Anat 2002;200:617–27
13. Ribeiro Mde C, Hirt L, Bogousslavsky J, Regli L, Badaut J. Time
course of Aquaporin expression after transient focal cerebral
ischemia in mice. J Neurosci Res 2006;83:1231– 40
14. Taniguchi M, Yamashita T, Kumura E, Tamatani M, Kobayashi
A, Yokawa T, Maruno M, Kato A, Ohnishi T, Kohmura E,
Tohyama M, Yoshimine T. Induction of aquaporin-4 water
channel mRNA after focal cerebral ischemia in rat. Brain Res
Mol Brain Res 2000;78:131–7
15. Manley GT, Fujimura M, Ma T, Noshita N, Filiz F, Bollen AW,
Chan P, Verkman AS. Aquaporin-4 deletion in mice reduces
brain edema after acute water intoxication and ischemic stroke.
Nat Med 2000;6:159 – 63
16. Manley GT, Binder DK, Papadopoulos MC, Verkman AS. New
insights into water transport and edema in the central nervous
system from phenotype analysis of aquaporin-4 null mice.
Neuroscience 2004;129:983–91
17. Papadopoulos MC, Manley GT, Krishna S, Verkman AS.
Aquaporin- 4 facilitates reabsorption of excess fluid in vasogenic brain edema. FASEB J 2004;18:1291–3
18. Longa EZ, Weinstein PR, Carlson S, Cummins R. Reversible
middle cerebral artery occlusion without craniectomy in rats.
Stroke 1989;20:84 –91
19. Zhou Y, Wei EQ, Fang SH, Chu LS, Wang ML, Zhang WP, Yu
GL, Ye YL, Lin SC, Chen Z. Spatio-temporal properties of
5-lipoxygenase expression and activation in the brain after focal
cerebral ischemia in rats. Life Sci 2006;79:1645–56
20. Joshi CN, Jain SK, Murthy PS. An optimized triphenyltetrazolium chloride method for identification of cerebral infarcts.
Brain Res Brain Res Protoc 2004;13:11–17
21. Lin TN, He YY, Wu G, Khan M, Hsu CY. Effect of brain edema
on infarct volume in a focal cerebral ischemia model in rats.
Stroke 1993;24:117–21
22. Zhan C, Yang J. Protective effects of isoliquiritigenin in transient
middle cerebral artery occlusion-induced focal cerevral ischemia in rats. Pharmacol Res 2006;53:303–9
23. Yokota C, Kaji T, Kuge Y, Inoue H, Tamaki N, Minematsu K.
Temporal and topographic profiles of cyclooxygenase-2 expression during 24 h of focal brain ishemia in rats. Neurosci Lett
2004;357:219 –22
24. Paczynski RP, Venkatesan R, Diringer MN, He YY, Hsu CY, Lin
W. Effects of fluid management on edema volume and midline
shift in a rat model of ischemic stroke. Stroke 2000;31:1702– 8
25. Gueniau C, Oberlander C. The kappa opioid agonist niravoline
decreases brain edema in the mouse middle cerebral artery
occlusion model of stroke. J Pharmacol Exp Ther 1997;282:1– 6
26. Amiry-Moghaddam M, Otsuka T, Hurn PD, Traystman RJ,
Haug FM, Froehner SC, Adams ME, Neely JD, Agre P, Ottersen
OP, Bharedwei A. An alpha-syntrophin-dependent pool of
AQP4 in astroglial end-feet confers bidirectional water flow
between blood and brain. Proc Natl Acad Sci USA 2003;100:
2106 –11
27. Rosenbeg GA, Estrada EY, Dencoff JE. Matrix metalloproteinases and TIMPs are associated with blood-brain barrier opening
after reperfusion in rat brain. Stroke 1998;29:2189 –95
28. Yu GL, Wei EQ, Zhang SH, Xu HM, Chu LS, Zhang WP, Zhang
Q, Chen Z, Mei RH, Zhao MH. Montelukast, a cysteinyl
leukotriene receptor-1 antagonist, dose- and time-dependently
protects against focal cerebral ischemia in mice. Pharmacology
2005;73:31– 40
29. Gunnarson E, Zelenina M, Aperia A. Regulation of brain
aquaporins. Neuroscience 2004;129:947–55
© 2008 International Anesthesia Research Society
30. Kleindienst A, Fazzina G, Amorini AM, Dunbar JG, Glisson R,
Marmarou A. Modulation of AQP4 expression by the protein
kinase C activator, phorbol myristate acetate, decreases
ischemia-induced brain edema. Acta Neurochir Suppl
31. Hemmings HC Jr, Adamo AI, Hoffman MM. Biochemical
characterization of the stimulatory effects of halothane and
propofol on purified brain protein kinase C. Anesth Analg
1995;81:1216 –22
32. Han BC, Koh SB, Lee EY, Seong YH. Regional difference of
glutamate-induced swelling in cultured rat brain astrocytes. Life
Sci 2004;76:573– 83
33. Fan Y, Zhang J, Sun XL, Gao L, Zeng XN, Ding JH, Cao C, Niu
L, Hu G. Sex and region- specific alterations of basal aminoacid
and monoamino in the brain of AQP4 knock– out mice. J Neurosci Res 2005;82:458 – 64
34. Yamakura T, Sakimura K, Shimoji K, Mishina M. Effects of
propofol on various AMPA-, kainate- and NMDA-selective
glutamate receptor channels expressed in Xenopus oocytes.
Neurosci Lett 1995;188:187–90
35. Lingamaneni R, Birch ML, Hemmings HC Jr. Widespread
inhibition of sodium channel– dependent glutamate release
from isolated nerve terminals by isoflurane and propofol.
Anesthesiology 2001;95:1460 – 6
36. Sitar SM, Hanifi-Moghaddam P, Gelb A, Cechetto DF,
Siushansian R, Wilson JX. Propofol prevents peroxide-induced
inhibition of glutamate transport in cultured astrocytes. Anesthesiology 1999;90:1446 –53
37. Ridenour TR, Warner DS, Todd MM, Gionet TX. Comparative
effects of propofol and halothane on outcome from temporary
middle cerebral artery occlusion in the rat. Anesthesiology
38. Lee Y, Chung C, Oh YS. Effectiveness of porpofol pretreatment on the extent of deranged cerebral mitochondrial oxidative enzyme system after incomplete forebrain ischemia/
reperfusion in rats. J Korean Med Sci 2000;15:627–30
39. Nedelmann M, Wilhelm-Schwenkmezger T, Alessandri B,
Heimann A, Schneider F, Eicke BM, Dieterich M, Kempski O.
Cerebral embolic ischemia in rats: correlation of stroke severity
and functional deficit as important outcome parameter. Brain
Res 2007;1130:188 –96
40. Zausinger S, Hungerhuber E, Baethmann A, Reulen H, SchmidElsaesser R. Neurological impairment in rats after transient
middle cerebral artery occlusion: a comparative study under
various treatment paradigms. Brain Res 2000;863:94 –105
41. Busto R, Dietrich WD, Globus MY, Valdés I, Scheinberg P,
Ginsberg MD. Small differences in inraischemic brain temperature critically determine the extent of ischemic neuronal injury.
J Cereb Blood Flow Metab 1987;7:729 –38
Propofol Lessens AQP-4 Over-Expression and Cerebral Edema