Mild Hypothermia Attenuates Mitochondrial Oxidative Stress by Protecting Respiratory Enzymes and Upregulating MnSOD in a Pig Model of Cardiac Arrest
et al. (2012) Mild Hypothermia Attenuates Mitochondrial Oxidative Stress by Protecting Respiratory Enzymes
and Upregulating MnSOD in a Pig Model of Cardiac Arrest. PLoS ONE 7(4): e35313. doi:10.1371/journal.pone.0035313
Mild Hypothermia Attenuates Mitochondrial Oxidative Stress by Protecting Respiratory Enzymes and Upregulating MnSOD in a Pig Model of Cardiac Arrest
Ping Gong 0
Chun-Sheng Li 0
Rong Hua 0
Hong Zhao 0
Zi-Ren Tang 0
Xue Mei 0
Ming-Yue Zhang 0
Juan Cui 0
Christian Schulz, Heart Center Munich, Germany
0 Department of Emergency, Beijing Chaoyang Hospital, Capital Medical University , Beijing , People's Republic of China
Mild hypothermia is the only effective treatment confirmed clinically to improve neurological outcomes for comatose patients with cardiac arrest. However, the underlying mechanism is not fully elucidated. In this study, our aim was to determine the effect of mild hypothermia on mitochondrial oxidative stress in the cerebral cortex. We intravascularly induced mild hypothermia (33uC), maintained this temperature for 12 h, and actively rewarmed in the inbred Chinese Wuzhishan minipigs successfully resuscitated after 8 min of untreated ventricular fibrillation. Cerebral samples were collected at 24 and 72 h following return of spontaneous circulation (ROSC). We found that mitochondrial malondialdehyde and protein carbonyl levels were significantly increased in the cerebral cortex in normothermic pigs even at 24 h after ROSC, whereas mild hypothermia attenuated this increase. Moreover, mild hypothermia attenuated the decrease in Complex I and Complex III (i.e., major sites of reactive oxygen species production) activities of the mitochondrial respiratory chain and increased antioxidant enzyme manganese superoxide dismutase (MnSOD) activity. This increase in MnSOD activity was consistent with the upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA and protein expressions, and with the increase of Nrf2 nuclear translocation in normothermic pigs at 24 and 72 h following ROSC, whereas mild hypothermia enhanced these tendencies. Thus, our findings indicate that mild hypothermia attenuates mitochondrial oxidative stress in the cerebral cortex, which may be associated with reduced impairment of mitochondrial respiratory chain enzymes, and enhancement of MnSOD activity and expression via Nrf2 activation.
Recently, despite improvements in resuscitation techniques, the
survival rate in patients with cardiac arrest has not improved
[1,2,3], and adverse neurological outcomes remain a leading
problem following return of spontaneous circulation (ROSC),
which is closely related to a high post-resuscitative mortality and
poor quality of life [4,5]. One reason for these poor outcomes is
a lack of neuroprotective medications capable of ameliorating
ischemia/reperfusion (I/R) injury during the post-resuscitation
period. However, recently, an effective treatment that has been
confirmed clinically to improve neurological outcomes is mild
To data, the maximum therapeutic effect and mechanism of
action of mild hypothermia remains unclear. This may be due to
the complex mechanisms involved during global brain I/R injury
when triggered by cardiac arrest and resuscitation. Increasing
evidence has shown that the pathogenesis of post-resuscitation
brain injury is complicated by a complex cascade of processes such
as oxidative stress, excitotoxicity, disrupted calcium homeostasis,
pathological protease cascades and activation of cell death
signaling pathways, which are activated within minutes to hours
after injury, and continue for up to 72 h or longer . These
processes are temperature dependent i.e. may increase during
fever and can be inhibited by mild hypothermia .
Mitochondrial dysfunction and oxidative stress are considered
to be key determinants with respect to the extent of injury during
cerebral ischemia . Impairment of mitochondrial function leads
to reduced ATP production, impaired calcium buffering and, in
particular, the overproduction of reactive oxygen species (ROS).
Under physiologic conditions, ROS do not cause injury because
they are quickly scavenged by the intramitochondrial antioxidant
system, which includes antioxidants such as glutathione, and
antioxidant enzymes such as manganese superoxide dismutase
(MnSOD) and catalase, which are regulated by nuclear factor
erythroid 2-related factor 2 (Nrf2), a key nuclear transcription
factor in maintaining redox balance . Under pathologic
conditions such as cerebral I/R, ROS excessively produce, and
react with nitric oxide (NO) to produce nitrogen species (RNS).
The products exceed the scavenging capacity of the endogenous
antioxidant system in mitochondria, and consequently oxidative
stress occurs. As a result, the excess production of mitochondrial
ROS and RNS can damage mitochondria by initiating
peroxidation of intramitochondrial lipids and proteins, inhibiting the
activity of mitochondrial respiratory enzymes and breaking
Figure 1. Experimental procedure. HT, mild hypothermia group; NT, normothermia group; BC, blank control group; VF, ventricular fibrillation;
CPR, cardiopulmonary resuscitation; ROSC, restoration of spontaneous circulation; NDS, neurologic deficit scores; F, female; M, male.
mitochondrial DNA , which induces cell apoptosis or necrosis
Animal and patient studies have indicated that the protective
effect of mild hypothermia against brain injury may be due to
a reduction in brain metabolism, inhibition of excitatory amino
acid release, attenuation of the immune response, or modification
of cell death signaling pathways [4,8,12,13,14,15]. However, the
effect of mild hypothermia on mitochondrial oxidative stress and
the exact mechanisms involved in cardiac arrest patients remain
In the present study, we employed a swine model of cardiac
arrest to test the hypothesis that whole-body mild hypothermia
attenuates mitochondrial oxidative stress in cerebral cortex by
reducing impairment of mitochondrial respiratory chain enzymes
and enhancing the activity and expression of MnSOD via Nrf2
activation following ROSC.
Materials and Methods
This study was carried out in strict accordance with the
guideline for animal care and use established by the Capital
Medical University Animal Care and Use Committee. The
protocol was approved by the Committee on the Ethics of Animal
Experiments of Capital Medical University (Permit
Number:2010D-013). All surgery was performed under anesthesia and analgesia,
and all efforts were made to minimize suffering.
Twenty-one male and 16 female, inbred Chinese Wuzhishan
minipigs [Permit Number: SYXK (Beijing) 2008-0007, the
Institute of Animal Sciences, Chinese Academy of Agricultural
Sciences, Beijing, China] aged 46 months, weighing
24.561.7 kg, were fasted overnight, but had free access to water.
Our choice for the Wuzhishan minipig is due to their
characteristics similar to human beings in the histologic structures
and physiology, and especially due to the highest inbreeding
coefficient (more than 0.965), stable heredity and little variability
between individual animals after 20 generations of inbreeding
. Thus, this sort of inbred minipig is considered as more
appropriate experimental animal for cardiac arrest.
The animals were premedicated with an intramuscular injection
of ketamine (20 mg/kg), followed by cannulation of an ear vein
and intravenous administration of propofol (2 mg/kg) for
endotracheal intubation 10 min later. Anesthesia was maintained
by injection of sodium pentobarbital (8 mg/kg/h) and fentanyl
(5 mg/kg/h). After premedication, pigs were supinely secured on
the operating table and given normal saline (10 mL/kg/h)
through a vein to maintain a central venous pressure of 5
12 mmHg. After endotracheal intubation, the animals were
ventilated with a volume-controlled ventilator (Servo 900c,
Siemens, Munich, Germany) with a tidal volume of 15 mL/kg,
FiO2 of 0.21, and ventilation rate of 12 to 20 breaths/min.
Endtidal PCO2 was monitored with an in-line infrared capnograph
placed in the airway. Ventilation rate and tidal volume were
adjusted to maintain normocapnia (3545 mmHg). Arterial blood
gases (ABL80, Radiometer, Copenhagen, Denmark) were
analyzed to confirm adequate baseline ventilation.
The thorax skin was shaved to secure standard lead II
electrocardiogram surface electrodes. To measure aortic pressure,
a fluid filled catheter was advanced from the left femoral artery
into the thoracic aorta. To measure right atrial pressure,
pulmonary artery wedge pressure and cardiac output, a
SwanGanz catheter (7-Fr, Edwards Life Sciences, Irvine, California,
USA) was advanced from the left femoral vein and flow-directed
into the pulmonary artery. The Electrocardiograph (ECG), the
aortic, right atrial and pulmonary artery wedge pressure, and
cardiac output were monitored continuously throughout the
experiment with a monitor (Vigilance II, Edwards Life Sciences,
Irvine, California, USA). To induce ventricular fibrillation (VF),
a 5-Fr pacing catheter was advanced from the right femoral vein
into the right ventricle. To cool pigs, a central venous catheter
(IcyTM, Alsius Corp., Irvine, CA, USA) was advanced from the
right external jugular vein into the superior vena cava. A
temperature sensing Foley catheter (Integral Medical Products
Co., Ltd, Shaoxing, China) was inserted into the bladder following
fistulation. The central venous catheter and the temperature
sensing Foley catheter were connected to an external cooling
device (CoolGard 3000 system, Alsius Corp., Irvine, CA, USA).
The operation was performed using aseptic surgical techniques.
All catheters were calibrated before use, and unfractionated
heparin (100 U/kg) was administered to prevent the catheter from
After instrumentation, thirty minutes were allowed for
hemodynamic stabilization. During the period, intra-bladder
temperature was adjusted to 37uC using a heating lamp and warm packs,
or an electric fan and ice bags. VF was induced by programmed
electric stimulation through the fibrillation catheter, and was
confirmed by the VF wave in the ECG and sharply decreased
blood pressure (Figure 1). Once VF occurred, mechanical
ventilation was discontinued. After 8 min of untreated VF, CPR
was started with chest compressions performed by the same
investigator, an experienced CPR technician from our laboratory.
Meanwhile, ventilation was conducted using a bag respirator
attached to the endotracheal tube with room air, and the
compression-to-ventilation ratio was 30:2. After 2 min of CPR,
a single 150 J biphasic electrical shock was attempted with a Smart
Biphasic defibrillator (Philips Medical Systems, Andover, MA,
USA). If VF still persisted, another 2-min of CPR was resumed,
followed by the first bolus of epinephrine (30 mg/kg) via the
femoral vein. Additional doses of epinephrine were administered,
if needed, every 3 min until ROSC was achieved. Two hundred J
was used for the second and all subsequent attempts. We defined
ROSC as an organized cardiac rhythm with a mean aortic
pressure of greater than 60 mmHg, continuously sustained for at
least 10 min or more. Resuscitation procedures were terminated if
animals had no ROSC after 20 min of CPR.
After ROSC, pigs were randomized into mild hypothermia
group (HT, n = 16), or normothermia group (NT, n = 16) subjected
to the same treatment as mild hypothermia group with the
exception to no cooling. Another 5 pigs were for the blank control
group (BC, n = 5), which only received the same anesthesia and
surgery as other groups without induced VF and mild
hypothermia. Pigs in the normothermic and mild hypothermic groups were
again randomly assigned to subgroups on the basis of the time of
euthanasia, namely normothermia 24 h after ROSC (NT-24 h,
n = 8), mild hypothermia 24 h after ROSC (HT-24 h, n = 8),
normothermia 72 h after ROSC (NT-72 h, n = 8), and mild
hypothermia 72 h after ROSC (HT-72 h, n = 8; Figure 1).
Immediately after ROSC, mechanical ventilation was resumed
with the same setting as the one used before the induction of VF.
Meanwhile, according to the landmark study by Bernard et al.
animals were actively cooled to a target body temperature of 33uC
(1.0uC/h), maintained at this temperature for 12 h, and then
actively rewarmed (0.5uC/h) to 37uC using a CoolGard 3000
system. During the induction and maintenance of mild
hypothermia, all animals received pancuronium bromide (0.1 mg/kg) IV to
prevent shivering and muscle movement. Administration was
repeated if needed.
After ROSC, pigs received standardized post-resuscitative
intensive care till the end of the rewarming phase, and then were
returned into their cages. The room temperature was maintained
Values are mean 6 SD. *p,0.01 vs. NT group 12 h after ROSC. HT, mild
hypothermia group; NT, normothermia group; BC, blank control group; ROSC,
restoration of spontaneous circulation; bpm, beats per minute; MAP, mean
aortic pressure; CO, cardiac output; PAWP, pulmonary artery wedge pressure;
CPP, coronary perfusion pressure.
between 20uC and 24uC. Hemodynamics were obtained hourly
during the observation period. At 24 or 72 h after ROSC, animals
were euthanized by injecting propofol (3 mg/kg) and then 10 mL
of potassium chloride (10 mol/L). The brain was immediately
removed by craniotomy and divided by a mid-sagittal cut. The
right hemisphere was dissected, and the hippocampus and
a portion of the precentral gyrus of the frontal lobe were fixed
in 4% buffered formalin for hematoxylin-eosin staining. Another
portion of the precentral gyrus of the frontal lobe was immediately
collected on ice for morphological examination by electron
microscopy. From the left hemisphere, frontal cortex samples (1
2 g) were excised, and the mitochondria were rapidly isolated to
test respiratory enzyme activity. The remaining mitochondria and
frontal cortex samples were snap-frozen in liquid nitrogen, and
stored at 280uC until used. These cerebral areas were chosen
because the cerebral cortex is closely related to functional
outcomes, and because the CA1 region is selectively vulnerable
to global cerebral ischemia in animal and human studies.
Isolation of mitochondria
Mitochondria were isolated from pig brain cortex according to
the protocol described previously  with minor modifications.
Briefly, before mitochondrial isolation, all solutions were cooled
below 0uC until the slight appearance of ice. Brain cortex tissues
from pigs were rapidly removed, immediately put into ice-cold
isolation medium (225 mmol/L mannitol, 75 mmol/L sucrose,
5 mmol/L HEPES, 1.0 mmol/L EGTA, 1 mg/mL defatted
bovine serum albumin, pH = 7.4) and shaken to wash out blood.
Brain tissues were minced on ice in 10 mL of ice-cold isolation
medium containing 0.05% (w/v) nagarse, and then manually
homogenized using a glass homogenizer. Thereafter, the
homogenate was added to 20 mL of ice-cold isolation medium and
centrifuged at 2,0006g at 4uC for 4 min. After centrifugation, the
supernatant was filtered using cheesecloth and centrifuged again at
12,0006g at 4uC for 9 min. To permeabilize synaptosomes, the
resulting pellet was dissolved with 10 mL of ice-cold isolation
medium containing 0.02% (w/v) digitonin, transferred to a small
glass homogenizer, and then homogenized manually. Finally, the
resultant homogenate was centrifuged at 12,0006g at 4uC for
11 min. After discarding the supernatant, the final pellet was
resuspended in 300 mL ice-cold isolation medium (20 mg protein/
mL) and kept on ice for further experimental assays.
Mitochondrial protein was determined using the Bradford method.
Determination of thiobarbituric acid reactive species
(TBARS) in mitochondria
Oxidative damage to biomembranes is characterized by lipid
peroxidation, which can be measured by the formation of TBARS
during an acid-heating reaction . The concentration of
TBARS is expressed as malondialdehyde (MDA) production. To
determine the status of mitochondrial oxidative stress, we
measured the levels of MDA in mitochondria using a commercial
MDA kit (GENMED, Shanghai, China), according to the
manufacturers instructions. Results were expressed as MDA
equivalents (nmol/mg mitochondrial protein). Each sample was
tested in triplicate.
Determination of protein carbonyls in mitochondria
Oxidative damage to protein is characterized by protein
carbonylation, measured based on the reaction with
dinitrophenylhydrazine. To determine the status of mitochondrial oxidative
stress, we measured the levels of protein carbonyls in mitochondria
using a commercial protein carbonyls kit (GENMED, Shanghai,
China), according to the manufacturers instructions. The
concentration of protein carbonyls was expressed as nmol/mg
mitochondrial protein. Each sample was tested in triplicate.
Assay for citrate synthase and MnSOD enzymatic activity
Citrate synthase is widely used as a mitochondrial marker
because its activity  and mRNA  are constitutively
expressed and do not change with age or pathological condition.
The assay for citrate synthase enzymatic activity was performed
spectrophotometrically as described previously [21,22,23].
MnSOD activity was measured spectrophotometrically at
550 nm using the MnSOD activity commercial kit (Nanjing
Jiancheng Bioengineering Institute, Nanjing, China), according to
the manufacturers instructions. We added KCN (3 mmol/L) into
mitochondrial lysates to inactivate residual Cu/ZnSOD and
extracellular SOD before performing assays, resulting in the
detection of only MnSOD activity. The enzymatic activity of
MnSOD was expressed as a ratio of citrate synthase activity. Each
sample was tested in triplicate.
Assay for respiratory enzymes activity
Mitochondria were lysed using the animal tissue mitochondria
lysis kit (GENMED, Shanghai, China), and the activities of
Complex I and Complex III in the mitochondrial respiratory
chain were measured using the animal mitochondrial respiratory
chain complexes I and Complex III activities quantitative
determination kit (GENMED, Shanghai, China). Complex I activity
was calculated as the rotenone-specific activity subtracted from the
total activity. The above was performed at 4uC. The activities of
mitochondrial respiratory chain complexes were expressed as
Figure 4. Activities of respiratory enzymes and MnSOD in mitochondria isolated from the swine frontal cortex. Respiratory enzyme (A
and B) and MnSOD (C) activities are expressed as a ratio of citrate synthase activity (mean 6 SD). *p,0.05 vs. BC group; #p,0.01 vs. NT group. HT,
mild hypothermia group; NT, normothermia group; BC, blank control group; ROSC, restoration of spontaneous circulation. CS, citrate synthase.
Western blotting analysis of MnSOD and Nrf2
Cytoplasmic and nuclear protein fractions were prepared using
NE-PER nuclear and cytoplasmic extraction reagents (Pierce
Biotechnology, Inc, Rockford, IL, USA) based on the
manufacturers instruction. Protein concentration of cell lysates was
determined using the Bradford method. Aliquots of protein
(30 mg) of cytoplasmic, nuclear or mitochondrial fractions were
separated in 10% SDS-PAGE and transferred to nitrocellulose
membrane. After blocking with 5% nonfat milk in PBS containing
0.2% Tween-20, membranes were incubated at 4uC overnight
with primary antibody, including rabbit polyclonal anti-Nrf2
(1:300; Abcam, Cambridge, MA, USA), rabbit polyclonal
antiMnSOD (1:500; StressGen, Assay Designs, Inc., Ann Arbor, MI,
USA), mouse polyclonal anti-GAPDH
(glyceraldehyde-3-phosphate dehydrogenase) (1:1000; Abcam, Cambridge, MA, USA),
mouse monoclonal anti-Lamin B2 (1:300; Pierce Biotechnology,
Inc, Rockford, IL, USA), rabbit polyclonal anti-VDAC1
(voltagedependent anion channel)/Porin (1:600; Abcam, Cambridge, MA,
USA) and mouse polyclonal anti-b-actin (1:1000; Sigma-Aldrich,
St. Louis, MO, USA). Membranes were then incubated with
horseradish peroxidase-conjugated secondary antibodies
(SigmaAldrich, St. Louis, MO, USA) for 2h. ECL reagent (GE
Healthcare, Piscataway, NJ, USA) was used for protein detection.
We measured optical densities of the immunoreactive bands using
ImageJ software (National Institutes of Health, Bethesda, MD,
USA). MnSOD, and cytoplasmic, nuclear and total Nrf2 protein
levels were normalized to Porin, GAPDH, Lamin B2 and b-actin,
respectively, and presented as a ratio.
Real-time RT-PCR for expression of MnSOD and Nrf2
We extracted total RNA from frozen swine frontal cortex
samples with Trizol reagent (Invitrogen, Carlsbad, CA, USA),
followed by reverse transcription to generate first-strand cDNAs
using the QuantiTec reverse transcription kit (Tiangen Biotech,
Beijing, China), according to the manufacturers instructions. As
the swine cDNA sequences for Nrf2 were not available in
GenBank, we performed RT-PCR to obtain its partial coding
sequence in advance, using primer pairs (Nrf2,
GCATTTCACATCACAGTAGG-39) designed by OLIGO 4.0 program based on the
multialignment of ortholog sequences known in humans. The
partial sequence of Nrf2 is available on request. Subsequently, we
determined the expression of Nrf2 and MnSOD mRNAs by
realtime RT-PCR. The primer sequences for MnSOD (Sus scrofa)
were F-59- CTGGAAGCCATCAAACGC-39 and
R-59TGAAACCGAGCCAACCC-39, and the primer sequences for
the endogenous reference b-actin (Sus scrofa) were
F-59TGGGTATGGGTCAGAAAG-39 and R-59-
CTCGTTGTAGAAGGTGTG-39. We used the Quant script RT Kit (Tiangen
Biotech, Beijing, China) for real-time RT-PCR analysis. The cycle
threshold (Ct) values of the interested genes were first normalized
with b-actin from the same sample, and then the relative
differences between the groups were calculated and expressed as
relative increases, setting the blank control group as 1. Each
sample was tested in triplicate.
Histopathology in the cortex and hippocampus
Tissue blocks (0.5 cm thick) from the cortex and hippocampus
were embedded in paraffin, sliced into 6 mm thick sections and
stained with hematoxylin and eosin (HE). In each microscopic
field at a magnification of 6400, the percentage of damaged
neurons was determined under a light microscope (Shanghai
zousun optical instrument Co., Ltd, Shanghai, China) by an
experienced doctor from the department of pathology in a blind
fashion. The criteria for neuronal cytopathology included an
eosinophilic cytoplasm, cytoplasmic vacuolation, perikaryal
shrinkage, and nuclear pyknosis . Three sections at least 15 mm
apart were examined for each pig and the mean value was taken.
Approximately 1 mm thick of cerebral cortex was sliced on ice,
immediately fixed overnight at 4uC with a 2.5% (v/v)
glutaraldehyde and 2.0% (w/v) paraformaldehyde, postfixed with 1% (v/v)
osmic acid for 2 h, and then dehydrated and embedded with
Epon812. The semi-thin sections were cut and stained with 1.0 %
(w/v) toluidine blue and observed under a light microscope
(Shanghai zousun optical instrument Co., Ltd, Shanghai, China).
Ultra-thin sections (approximately 4050 nm) were cut, double
stained with 2.0% (w/v) uranyl acetate and 2.0% (w/v) lead
citrate, and finally observed blindly under the HITACHI H-600
transmission electron microscope (Hitachi Scientific Instruments,
Mountain View, CA, USA).
To quantitatively assess the ultrastructural changes of
mitochondria, we used the morphometric analysis described previously
 with minor modifications. Briefly, at least four different
electron microscopic micrographs representing different areas in
Figure 5. Expressions of MnSOD, and cytoplasmic, nuclear and total Nrf2 protein in the swine frontal cortex. The expressions of
MnSOD (A) and Nrf2 (B) protein were measured by western blotting (left) using appropriate antibodies in the BC (n = 5), NT-24 h (n = 6), HT-24 h
(n = 7), NT-72 h (n = 5) and HT-72 h (n = 7) groups. The OD of each band was measured using Image J software, and the values of MnSOD, and
cytoplasmic, nuclear and total Nrf2 protein were normalized to Porin, GAPDH, Lamin B2 and b-actin, respectively (right). Data are expressed as the
mean 6 SD. *p,0.05 vs. BC group; #p,0.05 vs. NT-24 h group. HT, mild hypothermia group; NT, normothermia group; BC, blank control group;
ROSC, restoration of spontaneous circulation; OD, optical density.
an ultra-thin section from each pig were selected, and the mean of
these scores from the same ultra-thin section was regarded as the
score of a pig. The criteria for scoring were as follows: a score from
two to zero was given, where two was normal/not damaged, one
was influenced, and zero was bad. Both the matrix and the
membrane cristae were evaluated. Mitochondria displaying
a homogenous density in the matrix were scored as two.
Welldefined, tightly connected membrane cristae of mitochondria were
also scored as two. Thus, a maximum score of four and a minimum
score of zero could be obtained for mitochondria.
Neurological deficit scores (NDS)
We adopted NDS to evaluate the outcome of neurologic
function in pigs at 24 and 72 h following ROSC. NDS included
the levels of consciousness, motor and sensory function, respiratory
pattern, as well as behavior. The scores from each category were
summed. A minimum score of zero indicated that animals were
normal, whereas a total score of 400 represented brain death .
Investigators were blinded to the pigs respective treatments.
All data are presented as mean 6 standard deviation (SD)
except for NDS and ultrastructural scoring of mitochondria, which
are expressed as median and range. Three-way ANOVA (factors:
temperature, time, and gender) was used to assess overall
differences among groups for each of the variables, followed by
Bonferroni test for multiple comparisons. Levenes test for equality
of variance was used to indicate the multiple-comparison
procedure to be used. Survival rates were compared using Fishers
exact test. Ultrastructural scoring of mitochondria was statistically
analyzed using nonparametric Mann-Whitney U-test. Differences
were considered statistically significant when the p value ,0.05.
Analysis was performed using the software package SPSS 16.0
(SPSS Inc., Chicago, Illinois, USA).
Physiologic, hemodynamic and resuscitation data
There was no significant difference in the number of male or
female pigs in each group (Figure 1). At baseline, 24 h or 72 h
after ROSC, weight, temperature, arterial lactate and PaO2, as
well as hemodynamic parameters, were similar for pigs in all
groups. Twelve hours after ROSC, heart rate and cardiac output
(CO) were significantly decreased in the HT group compared with
the NT group (p,0.01). CPR time was not statistically different
between the NT and HT groups (3.361.2 vs. 3.461.3 min,
respectively; Table 1).
Survival rate and neurological outcome
We successfully resuscitated 32 pigs. Following ROSC, 6 out of
8 pigs in the NT-24 h, 7 out of 8 in the HT-24 h, 5 out of 8 in the
NT-72 h, and 7 out of 8 in the NT-72 h survived (Figure 1).
Although there was a trend towards higher survival rates at 24 and
72 h after ROSC in the HT group compared with the NT group
(87.5% vs. 75.0%, 87.5% vs. 62.5%, respectively), the difference
did not reach statistical significance (p = 0.60, p = 0.32,
respectively). Seven pigs (3 females, 4 males) died due to hemodynamic
instability during the post-resuscitation period. In the NT or HT
group, the differences in total survival rate between the females
and the males were not statistically significant (p = 0.68, p = 0.48,
We found that the NDS were significantly lower in the HT
group when compared with the NT group at 24 h and 72 h after
ROSC (p,0.01; Figure 2), indicating mild hypothermia could
improve neurological outcome. In addition, the NDS for female
pigs were slightly lower than for male pigs at 24 h and 72 h after
ROSC in the NT or HT group, but this difference was not
statistically significant (all P.0.05).
Effect of mild hypothermia on mitochondrial lipid and
To evaluate oxidative stress in mitochondria isolated from the
pig cerebral cortex at 24 h and 72 h after ROSC and determine
the effect of mild hypothermia, we first examined the extent of
lipid and protein peroxidation in mitochondria. MDA and protein
carbonyls were significantly increased in brain mitochondria from
normothermic and mild hypothermic pigs at 24 h after ROSC
when compared to blank control pigs (p,0.01), whereas the
increase was attenuated in the HT group when compared with the
NT group (p,0.01; Figures 3A and 3B). Seventy-two hours after
ROSC, MDA and protein carbonyl levels almost returned to
baseline levels, but there was no difference between the NT and
HT groups (Figures 3A and 3B). Moreover, in the NT or HT
group MDA and protein carbonyl levels for the female pigs
showed a slight decrease when compared to the male pigs (all
Effect of mild hypothermia on respiratory enzymes
Complex I and Complex III activity are significantly decreased
at 24 h after ROSC (p,0.01), and were restored at 72 h after
ROSC (Figures 4A and 4B). However, mild hypothermia
attenuated this decrease (p,0.01), indicating its ability to reduce
impairment of respiratory enzymes and thus reduce the
production of ROS. In the NT or HT group, the differences in
Complex I and Complex III activity between females and males
all had no statistical significance (all P.0.05).
Effects of mild hypothermia on MnSOD activity, and
MnSOD mRNA and protein expressions
To determine the effects of mild hypothermia on the activity of
MnSOD, and on the expression of MnSOD mRNA and protein
in the pig cerebral frontal cortex at 24 h and 72 h following
ROSC, we examined enzymatic activity spectrophotometrically,
protein levels using western blotting analysis and mRNA levels
using real-time PCR. We observed a significant increase in
MnSOD activity (p,0.01; Figure 4C), and a consistent increase in
MnSOD protein and mRNA expression caused by global brain I/
R in the NT-24 h group when compared with the BC group.
Interestingly, high MnSOD activity, upregulated expression of
MnSOD protein and mRNA in the HT group was observed when
Figure 7. Representative microscopic changes in the cortex and hippocampus. Hematoxylin and eosin stained sections (6200), in the
precentral gyrus of the frontal lobe (A and B) and CA1 area of the hippocampus (C and D) at 24 h following ROSC, showed changes in damaged
neurons, including an eosinophilic cytoplasm, loss of Nissl substance and nuclear pyknosis (arrows). The number of damaged neurons from
hypothermic pigs (B and D) was markedly decreased when compared to normothermic pigs (A and C).
compared with the NT group (Figures 4C, 5A and 6A). We also
found, at 72 h following ROSC, that MnSOD activity, and
MnSOD protein and mRNA expression, were slightly increased in
the NT group, whereas there were no significant difference
between the HT and NT groups (Figures 4C, 5A and 6A).
Though we found that in the NT or HT group MnSOD activity
for female pigs was slightly high when compared to male pigs at
24 h and 72 h after ROSC, this difference did not have statistical
significance (all P.0.05). In each group, no significant gender
difference was found in MnSOD mRNA levels (all P.0.05).
Effects of mild hypothermia on Nrf2 protein and mRNA
expressions, and Nrf2 nuclear translocation
As mentioned before, Nrf2 plays a central role in maintaining
redox balance. The Nrf2-dependent cytoprotective pathway has
been shown to induce gene expression of antioxidant enzymes
including SOD. We therefore examined the effects of mild
hypothermia on Nrf2 nuclear translocation in the pig cerebral
frontal cortex. Western blotting showed that Nrf2 protein in the
nuclear fraction increased, whereas it decreased in the cytosolic
fraction at 24 h following ROSC in the NT group when compared
with the BC group (Figure 5B), indicating translocation of Nrf2
from the cytosol to the nucleus. In addition, we found that Nrf2
nuclear translocation was significantly elevated in the HT group
when compared with the NT group (Figure 5B).
To investigate the translational regulation of Nrf2 triggered by
oxidative stress, we next examined the expression of Nrf2 mRNA
in the cerebral frontal cortex in our pig cardiac arrest model using
real-time PCR. The results showed a significant increase in the
expression of Nrf2 mRNA in the NT group when compared with
the BC group, and higher Nrf2 mRNA expression in the HT
group when compared with the NT group (Figure 6B). Also, we
found that the increase in Nrf2 mRNA expression was consistent
with the change of total Nrf2 protein level.
Figure 8. Representative electron micrographs of mitochondria (6100,000). Mitochondria were isolated from the swine frontal cortex at
24 h following ROSC. A: normal mitochondria with intact membrane cristae and a smooth matrix in the blank control group. B: slightly damaged
mitochondria with basically normal cristae and a slightly damaged matrix in the hypothermia group. C: markedy deranged mitochondria with
disrupted cristae and a damaged matrix in the normothermia group. Scale bar = 500 mm.
Moreover, we found, at 72 h following ROSC, that Nrf2
protein in the nuclear fraction and Nrf2 mRNA were slightly
increased in the NT group, whereas there were no significant
differences between the HT and NT groups (Figures 5B and 6B).
In each group, no significant gender difference was found in
Nrf2 mRNA levels and optical density of bands for cytosolic and
nuclear Nrf2 (all P.0.05).
Effect of mild hypothermia on histopathology in the
cortex and hippocampus
HE staining showed that, compared to normothermia,
treatment with mild hypothermia significantly decreased the number of
damaged neurons (Figure 7) in the precentral gyrus of the frontal
lobe (45.3%64.2% vs 28.3%62.7%, P,0.01) and in the CA1
area of the hippocampus (63.4%65.2% vs 35.3%62.9%, P,0.01)
at 24 h following ROSC. Furthermore, in each group no
significant gender difference in the number of damaged neurons
in the precentral gyrus of the frontal lobe and the CA1 area of the
hippocampus was observed, however, the female pigs showed
a slightly decreased number of damaged neurons when compared
to the male pigs (all P.0.05).
Effect of mild hypothermia on mitochondrial
morphology determined by electron microscopy
At 24 h following ROSC, ultrastructural damage of
mitochondria in the cerebral cortex was reduced in the HT group when
compared to the NT group (Figure 8) with a significantly elevated
score (3.2 [2.54.0] vs1.5 [1.02.0], P = 0.001). There was no
significant difference between female and male pigs in each group
Our experiments demonstrate that after treatment with
wholebody mild hypothermia, (1) neurological outcome is improved, (2)
mitochondrial oxidative stress in the cerebral cortex is attenuated
even at 24 h following ROSC, (3) impairment of mitochondrial
respiratory chain enzymes (Complex I and Complex III) is
reduced, (4)increased activity of MnSOD is elevated, (5)
upregulated expressions of MnSOD protein and mRNA are
enhanced and (6) activated nuclear factor Nrf2 is enhanced at 24 h
and 72 h following ROSC.
To mimic the clinical application of mild hypothermia in
patients with cardiac arrest , by means of the CoolGard 3000
system, we intravascularly induced cooling, maintained mild
hypothermia (32uC34uC ) for 12 h, and then actively rewarmed
(0.5uC/h) in a well established swine cardiac arrest model induced
by VF [26,27,28,29,30,31,32,33]. The results showed a
significantly lower NDS in the HT group when compared with the NT
group at 24 h or 72 h after ROSC, indicating mild hypothermia
could improve neurological outcome. This is consistent with
previous evidence from animals and humans [1,4,6,7,8]. There
was a trend towards higher survival rates presented at 24 h and
72 h after ROSC, nevertheless the difference did not reach
statistical significance. Morphologically, we also observed that
mild hypothermia alleviated microscopic and ultrastructural
changes in the cerebral cortex caused by global I/R at 24 h after
ROSC (Figure 7 and 8).
The effect of hypothermia on oxidative stress is controversial.
Camara et al. found that hypothermia moderately enhanced
superoxide anion (O22) generation by mitochondria, and
markedly slowed O22 dismutation in the guinea pig isolated
perfused heart , suggesting that hypothermia may increase
oxidative stress. However, Lei et al. reported that mild
hypothermia decreased lipid peroxidation in the dog cerebral
cortex 2 h following ROSC . In this study, we demonstrated
that whole-body mild hypothermia attenuated mitochondrial
oxidative stress in the cerebral cortex even at 24 h following
ROSC, consistent with the result of Lei et al. The reasons for this
discrepancy are thought to include different animal models, and
different depth of hypothermia. Cold may be a sort of stress to
cells, and thus, lower temperatures could enhance oxidative stress.
In the above study by Camara et al., the magnitude of ROS
production was found to be inversely proportional to the
temperature change . Moreover, most enzyme activities
decreases by 50% for every 10uC fall in temperature . It is
likely that lower temperatures not only increase ROS generation
but also decrease removal of ROS due to the reduced activity of
enzymes responsible for scavenging ROS.
Any alteration in the balance between the generation and
removal of ROS is considered oxidative stress. To determine the
mechanisms underlying the attenuated oxidative stress by mild
hypothermia, we further investigate the generation and removal of
Possibly up to 90% of intracellular ROS are generated in the
mitochondrial electron transport chain (namely complexes I and
III) where electron leakage normally occurs . After ROSC,
especially in the early stage of post-ROSC, mitochondrial
oxidative phosphorylation is slowed down, resulting in reduced
generation of ATP, and thereby electrons are diverted to the
Qcycle through the electron transport chain, generating excessive
ROS. Mitochondrial electron transport chain per se could be
impaired by ROS at the same time of generating ROS. In this
experiment, we demonstrated that the activity of Complex I and
Complex III was significantly reduced in the cerebral cortex
mitochondria from normothermic pigs when compared to blank
control pigs not subjected to I/R injury, consistent with previous
studies [5,38]. Moreover, we found that the decreased activities of
Complex I and Complex III by I/R was significantly improved
after treatment with mild hypothermia, indicating mild
hypothermia could reduce impairment of respiratory enzymes. It was
reported that the rate of mitochondrial ROS generation is
inversely proportional to the rate of oxidative phosphorylation,
increasing when activity of mitochondrial respiratory enzymes are
inhibited [37,39]. We thereby speculate that the improved activity
of mitochondrial respiratory enzymes could attenuate the
overproduction of ROS even at 24 h following ROSC.
MnSOD is a nuclear encoded primary antioxidant enzyme
located exclusively in the matrix of the mitochondrion  and is
therefore a pivotal part of the antioxidant defense involved in
protecting against oxidative injury to mitochondria . We
found a significant increase in MnSOD activity caused by global
brain I/R in the NT-24 h group. To clarify the mechanisms
underlying the increased MnSOD activity observed in the cerebral
cortex from pigs following ROSC, we further examined the
change of the protein and mRNA levels of MnSOD, and found
that the upregulation in MnSOD protein and mRNA levels are
consistent with a higher activity of MnSOD in normothermic pigs,
whereas MnSOD protein and mRNA levels were upregulated
after rewarming in hypothermic pigs. Based on the presence of
protein and lipid peroxidation, we speculate that the enhanced
ROS generation could overwhelm the removal of ROS in the
cerebral cortex from pigs following ROSC, and that the increased
MnSOD activity and reduced ROS generation after treatment
with mild hypothermia could lead to a decrease in oxidative stress,
and thus protect against global brain I/R injury.
SOD is known to be regulated by Nrf2. Nrf2 is a basic leucine
zipper (bZIP) transcription factor that serves as a central regulator
of genes encoding a battery of antioxidant proteins and
electrophile enzymes . To our knowledge, it is found for the
first time that there was a translocation of Nrf2 to the nucleus from
the cytoplasm, which may result from oxidative stress, in the
cerebral cortex from normothermic pigs following ROSC, and
that this translocation of Nrf2 was consistent with an increase in
MnSOD protein and mRNA expression. Also, we found that there
was an increase in Nrf2 mRNA and protein expression in the
cerebral cortex from normothermic pigs following ROSC. More
importantly, these tendencies were enhanced after treatment with
Because of the difficulty in supplying inbred Chinese Wuzhishan
minipigs, we did not choose pigs of the same sex. In the present
study, analysis by gender showed that no significant gender
differences were found for post-resuscitative brain damage and on
mild hypothermic effects, although female pigs had a slightly
better outcome. However, increasing evidence demonstrates that
sexual hormonal differences due to sex dimorphism may be
implicated in the outcome of cerebral ischemia; in particular, the
neuroprotective effects of estrogen have been widely documented
in animals and humans [42,43,44,45]. There are several factors for
this discrepancy. First, in female animals, the estrous cycle affects
the outcome of ischemic brain injury . However, in our study,
all female pigs may have had low endogenous estrogen levels
because they were not in a state of estrus. Second, most of the pigs
(31/37) were sexually immature due to their age (,4 months old).
Finally, the small sample size of male and female pigs in each
group may have failed to test for potential differences. Although
there was no significant difference in gender among the control,
normothermic and hypothermic groups, gender difference may be
a potentially confounding factor.
However, our work has some limitations. First, to mimic the
unbroken process of whole-body mild hypothermia clinically
applied to cardiac arrest patients , we only aimed to the effects
after rewarming rather than during mild hypothermia. Oxidative
stress in mitochondria occur mainly in the early stages of
reperfusion, whereas it may last for dozens of hours even several
days due to the involvement of a myriad of secondary injury after
ROSC. Second, we did not concern with cytosolic copper-zinc
SOD (Cu/ZnSOD) and extracellular SOD (EsSOD), as well as
other antioxidants and antioxidant enzymes regulated by Nrf2,
e.g. GSH, catalase, which contribute to oxidative stress in
mitochondria, yet it is a early episode of ROS removal and a key
reaction that MnSOD catalyzes the dismutation of the superoxide
anion into oxygen and hydrogen peroxide .
In conclusion, our work has demonstrated that mitochondrial
oxidative stress of cerebral cortex is attenuated after treatment
with whole-body mild hypothermia even at 24 h following ROSC,
which may be one of the mechanisms underlying neurologic
protection. It appears that the effect of mild hypothermia on
mitochondrial oxidative stress is achieved, at least in part, by its
ability to reduce impairment of respiratory chain enzymes. Also,
we have demonstrated that the increased activity of MnSOD, and
the upregulated expression MnSOD protein and mRNA are
further enhanced after treatment with whole-body mild
hypothermia even at 24 h and 72 h following ROSC via the activation of
nuclear factor Nrf2, which may contribute to the attenuation of
mitochondrial oxidative stress.
The authors would like to thank Xian-Fei Ji, Zhi-Yu Su, Jun-Yuan Wu,
Shuo Wang for their technical assistance.
Conceived and designed the experiments: PG CSL. Performed the
experiments: PG HZ RH ZRT XM MYZ JC. Analyzed the data: PG.
Contributed reagents/materials/analysis tools: PG ZRT XM. Wrote the
paper: PG CSL.
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