Ischemic preconditioning or heat shock pretreatment ameliorates neuronal apoptosis following hypothermic circulatory arrest.
ABSTRACT Hypothermic circulatory arrest has been widely used in complex cardiac and aortic surgery. Stroke and/or neurologic injury can occur after prolonged hypothermic circulatory arrest, possibly due to apoptosis. Ischemic preconditioning has been widely used as a neuroprotective tool, but its application in neuronal injury under hypothermic circulatory arrest has never been studied.
Forty male New Zealand white rabbits were placed on closed-chest cardiopulmonary bypass, subjected to hypothermic circulatory arrest, and rewarmed to normothermia. Experimental groups were treated with heat shock or ischemic preconditioning before hypothermic circulatory arrest. Hippocampal CA1 neurons were analyzed histopathologically. Apoptosis was confirmed by TUNEL assay and Western blot analysis, and serum S-100beta levels, c-Fos and Bcl-2 antibodies, and caspase-3 and heat shock protein 70 levels were measured.
After 2-hour hypothermic circulatory arrest and 4-hour reperfusion, apoptosis was observed in hippocampal CA1 neurons with elevation of serum S-100beta levels, which could be ameliorated by ischemic preconditioning or heat shock manipulations. TUNEL-positive nuclear expression of caspase-3 increased after hypothermic circulatory arrest (3.08% +/- 0.71%, P <.001) and was diminished with ischemic preconditioning (1.61% +/- 0.42%) and heat shock (1.72% +/- 0.38%) manipulations. Ischemic preconditioning or heat shock manipulations produced diverse patterns of heat shock protein 70, c-Fos, and Bcl-2 protein expression, suggesting that these manipulations provide neuroprotection via different pathways.
Ischemic preconditioning and heat shock can attenuate hippocampal CA1 neuronal apoptosis after prolonged hypothermic circulatory arrest under cardiopulmonary bypass. The expression of heat shock protein 70 may not play a major role in the first window of ischemic preconditioning-induced neuroprotection.
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ABSTRACT: Four patients are reported in whom the aortic arch and variable portions of the ascending and descending aorta were replaced with a prosthesis. In three patients the preoperative diagnosis was dissecting aneurysm of the aortic arch and in one an arteriosclerotic aneurysm of the aortic arch was present. A combination of surface cooling and cardiopulmonary bypass was utilized to produce total body hypothermia. Arch replacement was carried out during a period of total circulatory arrest. Cardiopulmonary bypass was then utilized to warm the patient and resuscitate the heart. The average duration of cerebral ischemia was 43 minutes and the average duration of myocardial ischemia was 74 minutes. The average lowest esophageal temperature was 14 degrees C., and the average lowest rectal temperature was 18 degrees C. Three patients are alive and well 4 to 13 months following surgery. One patient died 4 days postoperatively of pulmonary insufficiency. This experience indicates that by utilizing total body hypothermia and circulatory arrest aortic arch replacement can be carried out with an acceptable mortality rate. Corrective surgery could be offered to patients with life-threatening enlarging aneurysms of the aortic arch.Journal of Thoracic and Cardiovascular Surgery 01/1976; 70(6):1051-63. · 3.53 Impact Factor
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ABSTRACT: This study was undertaken to assess neurodevelopment of children after biventricular repair of congenital heart defects. Full-scale, performance, and verbal IQs of 69 patients who had undergone biventricular repair were assessed at 5 years of age with the Wechsler Preschool and Primary Scales of Intelligence-Revised. The Wide Range Assessment of Visual-Motor Abilities was used to measure visual-motor skills. Regression analyses adjusting for parental IQ and socioeconomic status were used to evaluate outcome predictors. Median age at repair was 91 days (range 1-1558 days). Hypothermic circulatory arrest was used in 35 cases (mean duration of hypothermic circulatory arrest 33 +/- 17 minutes). Mean full-scale, performance, and verbal IQs for the entire study population were within the reference range (full-scale 96.9 +/- 15.9, performance 96.6 +/- 16.8, verbal 97.7 +/- 15.2). Anatomic diagnosis, age at operation, and use of hypothermic circulatory arrest did not influence full-scale IQ (P =.66, P =.14, and P =.46, respectively), performance IQ (P =.64, P =.36, and P =.73, respectively), or verbal IQ (P =.74, P =.08, and P =.39, respectively). Among patients subjected to hypothermic circulatory arrest, duration of arrest was evaluated as a predictor of outcome. After adjustment for parental IQ, full-scale (P =.12), performance (P =.07), and verbal (P =.22) IQ scores of patients with more than 39 minutes of hypothermic circulatory arrest were not different from those of patients who had arrest periods of 39 minutes or less. After adjustment for socioeconomic status, however, full-scale (P =.05) and performance (P =.03) IQ scores were lower among patients who had more than 39 minutes of hypothermic circulatory arrest. After adjustment for either parental IQ or socioeconomic status, patients with more than 39 minutes of arrest had lower scores on Wide Range Assessment of Visual-Motor Abilities subtests of visual-motor and fine motor abilities and on several performance IQ subtests. IQs of patients who had undergone biventricular repair of congenital heart defects were within the reference range. However, hypothermic circulatory arrest for longer than 39 minutes was associated with deficits in visual-motor and fine motor skills and possibly in full-scale IQ.Journal of Thoracic and Cardiovascular Surgery 05/2002; 123(4):631-9. · 3.53 Impact Factor
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ABSTRACT: Hypothermic circulatory arrest (HCA) is used in surgery for aortic and congenital cardiac diseases. Although studies of the safety of HCA in animals have been carried out, the degree to which metabolism is suppressed in patients during hypothermia has been difficult to determine because of problems with serial measurements of cerebral blood flow in the clinical setting. To quantify the degree of metabolic suppression achieved by hypothermia, we studied 37 adults undergoing operations employing HCA. Cerebral blood flow was estimated using an ultrasonic flow probe on the left common carotid artery, and cerebral arteriovenous oxygen content differences were calculated from jugular venous bulb and arterial oxygen saturations. Cerebral metabolic rates while cooling were then ascertained. The temperature coefficient, Q10, which is the ratio of metabolic rates at temperatures 10 degrees C apart, was determined. The human cerebral Q10 was found to be 2.3. The cerebral metabolic rate is still 17% of baseline at 15 degrees C. If one assumes that cerebral blood flow can safely be interrupted for 5 min at 37 degrees C, and that cerebral metabolic suppression accounts for the protective effects of hypothermia, the predicted safe duration of HCA at 15 degrees C is only 29 min. The safe intervals calculated from measured cerebral oxygen consumption suggest that shorter intervals and lower temperatures than those currently used may be necessary to assure adequate cerebral protection during hypothermic circulatory arrest.The Annals of Thoracic Surgery 07/1999; 67(6):1895-9; discussion 1919-21. · 3.45 Impact Factor
Ischemic preconditioning or heat shock pretreatment
ameliorates neuronal apoptosis following hypothermic
Chi-Hsiao Yeh, MD
Yao-Chang Wang, MD
Yi-Cheng Wu, MD
Pyng Jing Lin, MD
Objective: Hypothermic circulatory arrest has been widely used in complex cardiac
and aortic surgery. Stroke and/or neurologic injury can occur after prolonged
hypothermic circulatory arrest, possibly due to apoptosis. Ischemic preconditioning
has been widely used as a neuroprotective tool, but its application in neuronal injury
under hypothermic circulatory arrest has never been studied.
Methods: Forty male New Zealand white rabbits were placed on closed-chest
cardiopulmonary bypass, subjected to hypothermic circulatory arrest, and rewarmed
to normothermia. Experimental groups were treated with heat shock or ischemic
preconditioning before hypothermic circulatory arrest. Hippocampal CA1 neurons
were analyzed histopathologically. Apoptosis was confirmed by TUNEL assay and
Western blot analysis, and serum S-100? levels, c-Fos and Bcl-2 antibodies, and
caspase-3 and heat shock protein 70 levels were measured.
Results: After 2-hour hypothermic circulatory arrest and 4-hour reperfusion, apo-
ptosis was observed in hippocampal CA1 neurons with elevation of serum S-100?
levels, which could be ameliorated by ischemic preconditioning or heat shock
manipulations. TUNEL-positive nuclear expression of caspase-3 increased after
hypothermic circulatory arrest (3.08% ? 0.71%, P ? .001) and was diminished with
ischemic preconditioning (1.61% ? 0.42%) and heat shock (1.72% ? 0.38%)
manipulations. Ischemic preconditioning or heat shock manipulations produced
diverse patterns of heat shock protein 70, c-Fos, and Bcl-2 protein expression,
suggesting that these manipulations provide neuroprotection via different pathways.
Conclusions: Ischemic preconditioning and heat shock can attenuate hippocampal
CA1 neuronal apoptosis after prolonged hypothermic circulatory arrest under car-
diopulmonary bypass. The expression of heat shock protein 70 may not play a major
role in the first window of ischemic preconditioning-induced neuroprotection.
with minor postoperative neurologic deficits. Therefore, the search continues for
other methods to increase the permissible period of HCA.
HCA-induced neurologic sequelae result from neuronal cell death, including
necrosis and apoptosis. The pathologic neuronal damage of HCA includes neuronal
ypothermic circulatory arrest (HCA) is frequently used for cere-
bral protection during aortic arch surgery and in cases of complex
congenital heart disease.1,2However, the main limitation of HCA
is time. At a brain temperature of 10°C to 15°C, the onset of
permanent neurologic injury is delayed 40 to 50 minutes.3Selec-
tive antegrade cerebral perfusion and retrograde cerebral perfu-
sion4are other protective methods used during aortic arch surgery but are associated
From the Division of Thoracic and Cardio-
vascular Surgery, Chang Gung Memorial
Hospital, Keelung, Taiwan.
This research was supported by grant
CMRP-1200 from the Chang Gung Memo-
rial Hospital, Taipei, Taiwan.
Received for publication July 9, 2003; re-
visions requested Nov 26, 2003; accepted
for publication Dec 11, 2003.
Address for reprints: Chi-Hsiao Yeh, MD,
Division of Thoracic and Cardiovascular
Surgery, Chang Gung Memorial Hospital,
222 Mai-Chin Road, Keelung, Taiwan 204
J Thorac Cardiovasc Surg 2004;128:203-10
Copyright © 2003 by The American Asso-
ciation for Thoracic Surgery
Yeh et alCardiopulmonary Support and Physiology
The Journal of Thoracic and Cardiovascular Surgery●Volume 128, Number 2203
necrosis of the hippocampus, neocortex, basal ganglia, and
cerebellum.5Apoptosis is an additional cause of post-HCA
neurologic injury6and may explain the learning and mem-
ory deficits and impaired intellectual development seen after
HCA. The implications of the selective destruction of the
CA1 region are not known and require further investigation.
Animal studies have shown that global forebrain isch-
emia leads to a selective and delayed degeneration of hip-
pocampal CA1 pyramidal neurons.7,8Preconditioning, a
potent endogenous protective mechanism, significantly at-
tenuates the selective vulnerability of neurons to ischemia.9
Thus, exposure to a brief, nondamaging period of ischemia
before an ischemic insult enhances the resistance of the
brain to neuronal damage, a phenomenon referred to as
ischemic preconditioning (IP).10,11
The triggering factors and effectors of IP have been
under intense investigation. The elucidation of endogenous
mechanisms of IP against ischemia may lead to new thera-
peutic strategies that enhance neuroprotection under HCA.
The acquisition of ischemic tolerance by IP has been related
to the induction of heat shock proteins (HSPs),12,13which
prevent the disruption of proteins and bind to abnormal
proteins until they are refolded or disintegrated.
We hypothesized that IP can attenuate the neuronal ap-
optosis that causes neuronal injury seen after HCA and
results in increased HSP production and that HSPs can
attenuate neuronal apoptosis after HCA. We sought to char-
acterize the nature of neuronal cell death after HCA to
determine whether IP or HSP reduces neuronal apoptosis in
a rabbit model.
Materials and Methods
Hypothermic Circulatory Arrest Using
Forty male New Zealand white rabbits (2.5-3.5 kg) were premed-
icated with ketamine (10 mg/kg intramuscularly), anesthetized
with sodium pentobarbital (30 mg/kg intravenously), and then
given intermittent boluses of pentobarbital (5 mg/kg) and diaze-
pam (5 mg) as needed. Animals were intubated and ventilated with
oxygen-enriched room air using a respirator. The left femoral
artery and vein were catheterized for blood pressure monitoring
and fluid administration, respectively. Nasopharyngeal and rectal
temperatures were monitored. After systemic heparinization (250
U/kg intravenously), the right femoral artery and vein were can-
nulated for closed-chest cardiopulmonary bypass (CPB), which
was instituted with a membrane oxygenator (Maxima Plus oxy-
genation system, Medtronic, Inc, Cardiopulmonary Division, Ana-
heim, Calif) at a flow rate at 50 mL/kg/min. Animals were surface
cooled (using ice bags around the head and a cooling blanket) to a
nasopharyngeal temperature of 18°C and CPB was terminated.
During CPB, mean arterial pressure was maintained around 40 to
60 mm Hg. Circulatory arrest was maintained for 2 hours at 18°C,
followed by the reinstitution of CPB and rewarming. At normo-
thermia (37°C), animals were weaned from CPB and decannu-
lated, and protamine was administered.
Rabbits were monitored for 4 hours after HCA and remained
anesthetized on a ventilator. Animals were killed humanely while
fully anesthetized and were perfused with warm saline solution
and/or 4% paraformaldehyde. All animals received humane care in
compliance with the “Guide for the Care and Use of Laboratory
Animals” (National Institutes of Health publication no. 85-23,
revised in 1985).
Rabbits were randomly divided into 4 groups (n ? 10) as follows.
Controls: body temperature during CPB was kept around 37°C and
HCA was not performed.
HS: nasopharyngeal temperature was elevated to 43°C for 15
minutes using an external blanket before CPB, and HCA was
performed for 2 hours.
PC: IP (3 cycles of 2.5-minute ischemia followed by 5-minute
reperfusion) was performed 2 hours before CPB; ischemia was
induced by clamping the 3 main branches of the aortic arch
accessed from the neck incision, and 2-hour HCA was per-
formed followed by 4-hour reperfusion.
NPC: HCA was performed for 2 hours without any other manip-
ulation after institution of CPB.
Serum S-100? Levels
Concentrations of serum S-100? were determined in mixed venous
blood samples at induction of anesthesia, after the completion of IP
or heat shock, 5 minutes before HCA, 10 minutes after resuming
CPB after 2-hour arrest, at rewarming to normothermia, and 4
hours after reperfusion. After centrifugation, the samples were
frozen to ?20°C for batch analysis. Serum S-100? levels were
measured using a luminescence immunoassay kit (Sangtec-100,
LIA-mat; Sangtec Medical AB, Bromma, Sweden) and expressed
as an increase-fold of S-100? levels above baseline values.
In Vitro Apoptosis Studies
Terminal deoxynucleotidyl transferase–mediated dUTP-biotin
nick-end labeling (TUNEL) was performed as previously de-
scribed.14Paraffin sections were affixed and deparaffinized. After
mounting, the sections were examined by light microscopy, and
labeled nuclei were easily identified from the unstained back-
ground. To quantify apoptosis, 500 nuclei in the CA1 pyramidal
cell layer were identified in 10 randomly selected ?400 high-
power fields per section. Apoptotic cell counts were expressed as
a percentage of the total number of nuclei counted.
Western Blot Analysis
Tissue levels of intact and cleaved caspase-3 and HSP70 were
determined by Western blot analysis as described previously.14
Membranes were blocked and probed with a monoclonal anti-
caspase-3 and anti-HSP-70 antibodies (Neomarkers, Inc, Fremont,
Calif) at 1:200 dilution for 2 hours. Primary antibody binding was
revealed using an anti-mouse peroxidase conjugate (Dako, Carpin-
teria, Calif) for 1 hour and the ECL chemiluminescent detection
system (Amersham Life Science). The immunoreactive bands
were quantified by digital densitometric imaging (Kodak 1D Im-
age Analysis software; Eastman Kodak Co, Rochester, NY).
Cardiopulmonary Support and Physiology Yeh et al
204The Journal of Thoracic and Cardiovascular Surgery●August 2004
The immunohistochemical procedure was performed with the pa-
rietal section including the hippocampus. The avidin-biotin perox-
idase method was used for free-floating sections. Commercially
available polyclonal antibodies for c-Fos (Santa Cruz Biotechnol-
ogy, Inc, Santa Cruz, Calif), monoclonal antibody for Bcl-2 (Neo-
markers), and monoclonal anti-caspase-3 and anti-HSP-70 anti-
bodies (Neomarkers) were used as primary antibodies. Each
section was incubated with a primary antibody overnight at 4°C. A
0.05-mol/L Tris-HCl buffer solution (pH 7.6) with 0.3% Triton-X
was used throughout the entire immunohistochemical procedure.
Data are expressed as mean ? standard error of the mean (SEM).
Data were entered into an Excel spreadsheet (Microsoft Corpora-
tion, Redmond, Wash) and analyzed using SPSS software version
8.0 (SPSS, Inc, Chicago, Ill). Differences among groups were
analyzed by 1-way analysis of variance followed by Tukey mul-
tiple comparison procedure.
Serum Level of S-100?
An average threefold increase in S-100? levels from base-
line was observed in control (3.7 ? 1.0–fold increase), PC
(4.2 ? 0.8–fold increase), and HS (2.9 ? 0.5–fold increase)
groups 4 hours after the end of CPB (P ? .02). Serum
S-100? concentration after 4-hour reperfusion was signifi-
cantly higher in the NPC group (13.3 ? 1.2–fold increase)
than in other groups (P ? .001). After IP manipulation,
serum S-100? levels were significantly higher in the PC
group than in other groups (P ? .001) but decreased to
control and HS group levels after 4-hour reperfusion, sug-
gesting that IP induced sublethal neuronal injury and pro-
vided neuroprotection. Increased serum S-100? levels in the
HS group remained low through the experiment (Figure 1).
Both HS and PC groups showed increases in serum S-100?
levels similar to controls after 4-hour reperfusion, suggest-
ing that both manipulations ameliorate neuronal damage to
the same level produced without HCA.
Assessment of Apoptotic Cell Death Following HCA
Apoptosis was detected using the TUNEL method, which
allows in situ labeling of intranucleosomal DNA breaks in
cell nuclei (Figure 2). As expected, sections from control
animals showed minimal apoptotic nuclei (0.69% ?
0.28%), whereas TUNEL-positive cells were observed in
experimental groups after HCA. HCA induced significant
(P ? .001) neuronal apoptosis in the CA1 neuronal layers
(1.72% ? 0.38%, 3.08% ? 0.71%, and 1.61% ? 0.42% of
apoptotic CA1 neuronal cells were identified in the HS,
NPC, and PC groups, respectively). The number of DNA
nick-end–labeled cells was significantly reduced in the hip-
pocampal CA1 layer in the HS and PC groups (P ? .001)
compared with the NPC group. IP or heat shock before
HCA significantly lessened neuronal apoptosis induced by
prolonged circulatory arrest.
HSP70 and Caspase-3
The induction of HSP70 and caspase-3 in the hippocampus
after HCA was determined by Western blot analysis (Figure
3, A and B). In the NPC group, HSP70 remained undetect-
able in hippocampal protein extracts after CPB. However,
manipulations with heat shock before 2-hour HCA induced
significantly more accumulation of HSP70 in hippocampal
CA1 cells in the HS group (2767 ? 241 U, P ? .001 vs all
3 groups). HSP70 expression observed 4 hours after HCA
Figure 1. Changes in serum S-100? levels. Baseline, Induction of anesthesia; management, after the completion of
IP or heat shock; on bypass, 5 minutes before HCA; arrest, 10 minutes after resuming CPB after 2-hour HCA;
rewarming, rewarming to normothermia; end, 4 hours after reperfusion. Asterisk denotes a statistically significant
increase (P < .05) in serum S-100? levels from control values. Data are mean ? SEM from 4 different experimental
Yeh et alCardiopulmonary Support and Physiology
The Journal of Thoracic and Cardiovascular Surgery●Volume 128, Number 2 205
was significantly lower in the NPC group (582 ? 62 U) than
in the PC (1408 ? 139 U) and control (1126 ? 141 U)
groups (P ? .001). Immunostaining with monoclonal anti-
bodies raised against HSP70 (Figure 4, C) and caspase-3
(Figure 4, D) was performed 4 hours after the HCA insult to
confirm Western blot results. Using digital densitometry,
the expression of caspase-3 protein 4 hours after HCA was
significantly higher in the NPC group (24127 ? 5590 U)
than in the control (13936 ? 1928 U, P ? .01), HS (15263
? 5589 U, P ? .03), or PC groups (12925 ? 4296 U, P ?
.01), a finding consistent with the TUNEL findings. Western
blotting showed that IP had neuroprotective effects on in-
hibition of caspase-3 activation and prevented hippocampal
CA1 neuronal apoptosis in a manner similar to the HS group
but without inducing the same HSP70 level, suggesting that
the mechanism of IP-induced hippocampal CA1 neuropro-
tection in HCA under CPB did not involve HSP.
Bcl-2 and c-Fos Expression
The induction of Bcl-2 and c-Fos proteins in the hippocam-
pus was examined after HCA using immunohistochemistry
(Figure 4, A and B). As expected, no Bcl-2 expression was
observed in hippocampal CA1 neurons (Figure 4, A) of
control and NPC animals. In contrast, the IP and heat shock
manipulations induced a strong Bcl-2 labeling of the CA1
neurons, comparable to that observed in hippocampal CA1
sections from control and NPC groups. c-Fos expression
was strong in the control, PC, and HS groups (Figure 4, B).
CPB induced an inflammatory response that increased the
expression of c-Fos protein, which further increased with IP
or heat shock manipulations and decreased with HCA.
The current results indicate that IP and heat shock may
improve cerebral outcome after prolonged HCA, confirming
the previously reported neuroprotective effect of IP.10,11
Shake and colleagues15reported the benefit of pharmaco-
logically induced IP on neuroprotection during HCA. Brain
injury during cardiac surgery under HCA has been attrib-
uted to the extreme manipulations of CPB flow rate and
HCA,16which exacerbate the severity of cerebral ischemia-
reperfusion injury associated with HCA. Although most of
the early imaging changes in the brain subsided within days
to weeks after surgery without major neurologic deficits,17
their consequences may be further potentiated in the pres-
ence of HCA-induced hypoxic-ischemic brain injury. De-
layed cell death via apoptotic pathways is of special interest
because of the potential to intercept this process.18
Increased levels of S-100? protein have been measured
after cardiac operations, stroke, and several other neurologic
disorders. This marker has been adopted for clinical use by
many cardiac surgeons, with the expectation that repeated
measurements could indicate brain injury postoperatively.19
In the present study, in response to ischemic brain insult,
S-100? levels increased significantly shortly after rewarm-
ing was initiated. Smaller increases in S-100? concentra-
tions occurred after reperfusion in IP-treated animals, which
supports the theory that better brain protection is present
IP, a phenomenon whereby a brief episode of sublethal
ischemia and other nonlethal stressors produce protection
against a subsequent detrimental ischemic insult, had been
described in the heart20and in the brain.21There are 2 forms
of IP, early and late. Early preconditioning occurs within the
first few hours of an ischemic insult and its mechanism is
thought to be related to the Na/K ATPase channel on the
inner mitochondrial membrane.15Late preconditioning, on
the other hand, occurs usually after 4 to 6 hours up to a
maximum effect at 24 hours in response to ischemia and had
been related to the induction of HSPs.10HSPs have been
classified as part of a larger family of proteins designated as
molecular chaperones, and in this capacity could provide
support to proteins and enzymes threatened by acute isch-
emia. Hyperthermia pretreatment of cells in culture can
induce heat tolerance,22probably via the induction of HSPs.
HSPs may act as molecular chaperones, preventing proteins
from aggregating or denaturing under conditions of physi-
ological stress.23The expression of HSPs may be involved
in a cascade of events leading to neuroprotection, depending
on the severity of the insult, the liability of the different
types of cells, and the availability of cochaperone pro-
teins.24However, induction of HSPs in hippocampal neu-
rons after afferent stimulation can closely parallel selective
neuronal injury, suggesting that the presence of HSPs indi-
cates potentially lethal cell stress and expression of HSPs
after cerebral ischemia may be related to the intensity of
ischemic stress.25In our experimental model, the ischemic
insult immediately preceded hypothermic CPB and HCA.
The hypothermia in this model is likely delaying the protein
synthesis, resulting even less HSP production. Besides, HSP
induction requires DNA transcription and translation to
Figure 2. Effect of the HCA-induced neuronal apoptosis. Symbols
denote a statistically significant increase (P < .05) compared
with the control (*) or NPC (†) group.
Cardiopulmonary Support and PhysiologyYeh et al
206 The Journal of Thoracic and Cardiovascular Surgery●August 2004
occur, a process requiring at least 4 hours. However, this
current study shows that HCA can induce hippocampal CA1
cell apoptosis, which could be ameliorated by IP or heat
shock manipulations. The significant difference in the pat-
tern of HSP70 expression between IP and HSP groups, in
this study using semi-quantitative methods, implies that
HSP70 might not be the major factor in the neuroprotection
mechanism of early window of IP under HCA.
HSP overexpression protects CA1 region of the hip-
pocampal neurons from global cerebral ischemia, in part by
increasing Bcl-2 expression.26The induction of immediate
early genes (IEGs) proteins, such as c-Fos, is closely asso-
ciated with the molecular process determining neuronal
survival after global cerebral ischemia, and HSPs have an
important role in neuronal survival when present at the
onset of the ischemic insult.27In response to cerebral isch-
emia, neurons in the central nervous system express c-Fos,
an IEG.28The functional significance of c-Fos expression
after ischemia is controversial. Prolonged c-Fos induction
can precede postischemic neuronal death, and c-Fos and its
gene products are involved in neuronal injury29and in the
induction of apoptotic genes that lead to cell death, though
its expression is essential for recovery from ischemia.30A
close correlation exists between prompt induction of c-Fos
and c-JUN with neuronal survival after ischemic stress, and
the induction of IEG proteins may be closely related to the
molecular process determining neuronal survival after
global cerebral ischemia, although the exact mechanism is
still uncertain.24A close correlation was also found between
prompt induction of c-Fos/c-JUN and the absence of per-
manent ischemic or postischemic damage.24The signifi-
cance of c-Fos protein expression underlies the fact that it
forms transcription factor-AP1 complex by dimerization
with Jun family protein and that it regulates late gene
expression, such as HSPs, basic fibroblast growth factor,
nerve growth factor, and neurotrophins,29which indicate
that the potentiation of c-Fos expression may be an under-
lying neuroprotective mechanism after ischemia. The cur-
rent data show that c-Fos protein is induced only after CPB
with circulatory arrest (not in the control group) and is
Figure 3. Western blot analyses of HSP70 (A) and caspase-3 (B) levels. In the NPC group, HSP70 levels were
significantly lower (*P < .05 vs control group; †P < .05 vs NPC group) and activated caspase-3 levels were
significantly higher (*P < .05 vs control group) compared with other groups.
Yeh et al Cardiopulmonary Support and Physiology
The Journal of Thoracic and Cardiovascular Surgery●Volume 128, Number 2 207
positively correlated with neuronal survival. However, c-
Fos expression was significantly inhibited with elevated
caspase-3 expression and occurrence of apoptosis of CA1
neurons in the NPC group. The decreased level of c-Fos
may result from a significant, synthesis-inhibiting ischemia-
reperfusion insult to the CA1 neuron.
The present study demonstrates that (1) CPB can induce
IEG protein expression with trivial neuronal damage; (2) IP
confers neuroprotection to hippocampal CA1 cells, which is
associated with amelioration of apoptosis under HCA; (3)
heat shock before global ischemia under HCA can signifi-
cantly inhibit neuronal damage in hippocampal CA1 cells;
and (4) IP activation before HCA is associated with only
mild HSP expression within the first window of protection,
suggesting that other factors play a major role in early phase
of preconditioning-induced neuroprotection. It is hoped that
these results contribute to the characterization of physiolog-
ical markers associated with enhanced neuronal recovery
following HCA and will help to develop more relevant
therapeutic approaches for cerebral protection.
Figure 4. Correlation between induction of c-Fos, Bcl-2, HSP70, and caspase-3 in the hippocampus after 2-hour
HCA and 4-hour reperfusion. In the vulnerable hippocampal CA1 region, induction of Bcl-2 (A), c-Fos (B), and HSP70
(C) was significant in the PC and HS groups, and significantly apoptotic neuronal damage was visualized as
expression of the caspase-3 proteins (D) in the NPC group.
Cardiopulmonary Support and Physiology Yeh et al
208The Journal of Thoracic and Cardiovascular Surgery●August 2004
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Figure 4. Cont’d. C and D. For legend see Figure 4.
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