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Introduction
A short period of ischemia is known
to increase tolerance of the brain to a subsequent prolonged
ischemia by reducing brain infarction and apoptosis, this phenomenon
is termed as ischemic preconditioning (IPC) or ischemic tolerance[1].
The neuroprotection of cerebral ischemic preconditioning is divided
into acute and delayed effects[2]. Delayed
neuroprotection occurs 24 h after IPC, then becomes manifest
thereafter and lasts up to 7 d.
Recently, it has been reported that
indomethacin, a nonselective inhibitor of cyclooxygenase (COX),
applied 1 h prior to lipopolysaccharide (LPS) treatment abolished
the LPS-induce delayed neuroprotection against focal cerebral
ischemia and reperfusion injury[3]. The results suggested
that PGs pathway or inflammatory pathway might be involved in
triggering delayed neuroprotection by LPS[3]. It has also
been reported that PGs increased to a high level in the brain
tissues within hours following a short period of ischemia[4].
However it is still unknown whether a rise of PGs in the brain is
involved in the delayed neuroprotection induced by IPC.
COX, also known as prostaglandin H2
synthase, is the rate-limiting enzyme in the metabolism of
arachidonic acid into prostanoids (PGs and thromboxanes). COX-2,
which is normally expressed in the neurons of the brain, is
inducible in response to mitogen, endotoxin, and cytokines. Previous
studies have demonstrated that focal cerebral ischemia induced
strongly expressed COX-2 in neurons in penumbra and contributed
significantly to enlargement of infarction[5]. Since IPC
limits infarction induced by ischemia and reperfusion injury, it may
prevent the up-regulation of COX-2 following focal cerebral ischemia
and reperfusion insult. Recently it was shown that IPC stimulus
reprogrammed the response of gene transcription to subsequent
ischemia and reperfusion insult in mice. It was found that COX-2
showed lower expression of mRNA by IPC using a microarray analysis[6].
In the present study, the PGs
pathway was examined to evaluate whether it is involved in
triggering cerebral IPC, and effects of IPC on COX-2 expression were
evaluated following focal cerebral ischemia and reperfusion.
Material and methods
Materials
Indomethcin (Sigma-Aldrich, St Louit, MO, USA) was dissolved in Me2SO.
The compound 2,3,7-triphenyltetrazolium chloride (TTC,
Sigma-Aldrich, St Louis, USA) was dissolved in saline.
TRI-REAGENT-LS extraction kit was purchased from Molecular Research
Center, Inc (Cincinnati, USA). RNasin, dNTP, oligo (dT) 18 primer,
and Taq DNA polymerase were obtained from Sangon
Biotechnology Co (Shanghai, China). M-MuLV reverse transcriptase was
from Fermentas Inc (Vilnius, Lithuania). The DNA Master SYBR Green I
was from Roche Co (Mannheim, Germany). BCA protein assay kit and ECL
chemiluminescence system were purchased from Pierce Co (Rockford,
USA). Goat Polyclonal antibody specific for COX-2 was from Santa
Cruz Biotechnology, Inc (Santa Cruz, USA, working concentration
1:500). Rat mono-antibody specific for beta-actin was from
Sigma-Aldrich (St Louis, USA, working concentration 1:5000).
Supersignal West Pico Trial Kit was from Pierce Co (Rockford, USA).
Animals and experiment design
Male Sprague-Dawley rats weighing 220-250 g (the Shanghai
Experimental Animal Center of Chinese Academy of Sciences) were used
in this study. The rats were randomly divided into six groups (n=8
each). In the sham group (sham), the left common carotid artery was
exposed, and the external carotid artery and its branches were
isolated and coagulated without middle cerebral artery occlusion (MCAO).
The rats of the ischemia and reperfusion group (I/R) were subjected
to MCAO (90 min) and reperfusion (24 h). For the IPC group (IPC),
IPC was applied 48 h before MCAO and reperfusion. For indomethacin
group 1 (Indo1+IPC+I/R), the rats were treated with indomethacin (3
mg/kg ip) 1 h prior to IPC, and MCAO and reperfusion was applied 48
h later. For indomethacin group 2 (Indo2+IPC+I/R), the rats were
treated with indomethacin (3 mg/kg ip) 1 h after IPC, and MCAO and
reperfusion were applied 48 h later. The last group was used to
exclude the effects of indomethacin itself on injuries of MCAO and
reperfusion. The rats were treated with indomethacin (3 mg/kg ip) 48
h before MCAO and reperfusion (Indo+I/R).
COX-2 mRNA and protein expression
Another series of experiments were performed. The rats were
divided into four groups (n=6 each). In the sham operation
group, the left common carotid artery was exposed, and the external
carotid artery and its branches were isolated and coagulated without
MCAO. The rats of the ischemia and reperfusion group were subjected
to MCAO (90 min) and reperfusion (24 h). In the third group, IPC was
applied 48 h before MCAO and reperfusion. In the last group, only
IPC was applied.
Model of IPC IPC was induced
as described previously with minor modification[7].
Briefly, the left common carotid artery was exposed, and the
external carotid artery and its branches were isolated and
coagulated. A branch of the left internal carotid artery (the left
pterygopal artery) was isolated and coagulated. The right common
carotid artery was exposed. Then a cannula was inserted into the
external carotid artery stump and advanced into the internal carotid
artery to infuse saline into the brain. The saline infusion rate was
12 mL/h, which was controlled by a Harvard apparatus compact
infusion pump (USA). A surgery artery nip was used to clamp the
right common carotid artery. To achieve marked neuroprotection, we
chose 48 h after IPC as the time interval to apply ischemia and
reperfusion insult.
Evaluation of infarct area
Coronal sections of the brain (2 mm thick) were cut and immersed in
a 1% solution of TTC. The stained slices were then fixed by
immersion in phosphate-buffered 10% formaldehyde. The infarct area
and hemispheric area of each section were photographed by a digital
camera and quantitated using the public domain image processing and
analysis program developed at the National Institute of Health, USA.
The infarct area was expressed as percentage to the contralateral
hemisphere.
Real-time reverse transcription
PCR The expression of RNAs was determined by quantitative
real-time RT-PCR using a Continuous Fluorescence Detector (MJ
Research Incorporated, DNA Engine Opticn 2) with the DNA Master SYBR
Green I. Animals were killed 24 h after reperfusion and their brains
removed. A 4-mm-thick coronal brain slice was cut at the levels of
the optic chiasm, and the infarcted cortex was dissected using the
corpus callosum as a ventral landmark[8]. Total RNA was
prepared from the samples with TRI-REAGENT-LS extraction kit
according to manufacture's instructions. Complementary DNA was
created from RNA using TrueScript MMLV reverse transcriptase and
oligo d(T)18 primers. A 0.2 µg amount of RNA was included
in each reaction in a total volume of 20 µL. The reaction was
performed at 42 ºC for 2 h. Thereafter the mix was diluted
five-fold, and 2 µL was added to the PCR reaction mixture to yield a
total volume of 20 µL. The COX-2 primers were forward, 5'-CCA TGT
CAA AAC CGT GGT GAA TG-3'; reverse: 5'-ATG GGA GTT GGG CAG TCA TCA
G-3', which result in a PCR product of 374 bp. The amplification
reaction consisted of 35 cycles of denaturation (94 ºC, 30 s),
annealing (64 ºC, 30 s), and elongation (72 ºC, 45 s). The beta-actin
primers were forward, 5'AAG ATG ACC CAG ATC ATG TT3'; reverse: 5'TTA
ATG TCA CGC ACG ATT T3', which resulted in a PCR product of 286 bp.
The amplification reaction consisted of 35 cycles of denaturation
(94 ºC, 30 s), annealing (56 ºC, 30 s), and elongation (72 ºC, 45
s). Cycle numbers (crossing points, when amplification starts its
exponential phase) were used for statistical analysis. The lower the
cycle number indicates the higher the amount of initial template.
The PCR products were verified by agarose gel electrophoresis.
Western blot analysis A
4-mm-thick coronal brain slice was cut at the levels of the optic
chiasm, and the infarcted cortex was dissected using the corpus
callosum as a ventral landmark[8]. The isolated cortex
was homogenized in ice-cold lysis buffer. The protein content was
determined by BCA protein assay. Equal amounts of protein per lane
(50 mg) were loaded onto an 8 % polyacrylamide gel and separated by
electrophoresis at 120 V. Proteins were then transferred to PVDF
membrane at 25 mA for 90 min, and the membrane was blocked with 5%
nonfat dry milk in 1X TBS, 0.1% Tween-20 at 25 ºC with gentle
shaking, overnight. The PVDF membrane was then incubated with a goat
polyclonal antibody specific for COX-2 (1:500) 1 h at 25 ºC followed
by horseradish peroxidase-conjugated secondary antibody (rabbit
anti-goat) for 1 h at 25 ºC. Antibody labeling was detected by
Supersignal West Pico Trial Kit. After being stripped, the same PVDF
membrane was incubated with mono-antibody specific for beta-actin
(1:5000) as an internal control. Western blot results were
quantified by density.
Data analysis All data were
expressed as mean¡ÀSD. Differences between different groups were
assessed by a one-way analysis of variance and Student-Newman-Keuls
test. A value of P<0.05 was considered statistically
significant.
Results
Protective effects of IPC
At 24 h after operation,
sham-operated rats did not show any cerebral tissue damage in TTC
staining (not shown). IPC reduced the infarct area by 63% as
compared with the MCAO alone group. Indomethacin (3 mg/kg ip), a
nonselective inhibitor of COX, applied 1 h prior to or 1 h after IPC
failed to affect these protective effects. Indomethacin itself had
no effect on infarction by ischemia and reperfusion injury (Figure
1).
Effects of IPC on COX-2 mRNA
expression In agreement with previous study, low levels of COX-2
PCR products were observed in the brain of sham-operated rats (to
achieve the crossing points, the cycle number 25.8¡À2.4 was needed).
After transient 90 min MCAO and 24 h reperfusion, COX-2 mRNA
expression was significantly up regulated (the cycle number
decreased to 19.9¡À1.9, P<0.01). IPC significantly prevented
the up-regulation of COX-2 mRNA expression following ischemia and
reperfusion injury (the cycle number decreased to 23.7¡À2.4, P<0.01),
while IPC itself had no effect on the expression of COX-2 mRNA 72 h
later (Figure 2).
Effects of IPC on COX-2 protein
expression COX-2 protein was expressed at a low level in the
brain of sham-operated rats (132¡À35). IPC itself had no effects on
expression of COX-2 protein 72 h later (135¡À32). Ischemia and
reperfusion markedly increased COX-2 protein expression (362¡À28,
P<0.01). IPC prevented the up regulation of COX-2 protein following
ischemia and reperfusion injury (225¡À21, P<0.01) (Figure 3).
Discussion
IPC is a fundamental adaptive
response of organisms against deleterious stress such as ischemia
and other severe stimuli. In fact, this phenomenon exists in most
organs such as brain, heart, skeletal muscle, lung, liver, stomach,
intestine, and kidney. IPC shares some common mechanisms in
different organs. The elements that constitute this molecular
cascade of delayed IPC can be conceptually subdivided into three
major components: (i) "triggers" or sensors, (ii) "effectors" or
mediators, and (iii) "transducers", signaling pathways that connect
these two groups of molecules[1,9]. Endogenous active
substances and bio-synthesized proteins have been suggested as
triggers and end effectors of brain IPC, respectively. Adenosine,
glutamate, oxygen free radical, and nitric oxide (NO) are these
candidate substances that trigger brain IPC[1].
PGs are endogenous active substances
that are suggested to be involved in IPC in the heart. It has been
reported that pretreatment with indomethacin attenuated PGs release
and abolished acute cardioprotection by IPC in
different animal models in vivo or in vitro[10,11].
Exogenous PGs perfusion in isolated hearts mimicked
cardioprotection induced by cardiac IPC[12]. The
mechanisms of cardio-protection mediated by PGs are mainly related
to the direct effects of PGs on the heart, including antagonism of
adenylyl cyclase, activation of ATP-sensitive potassium channels,
inhibition of calcium influx, and attenuation of neutrophil
infiltration[13]. However, there was no study on the role
of PGs in neuroprotection induced by IPC. A period of ischemia as
short as 5 min can induce an early increase in PGs levels after 2 h
of reperfusion in the brain[4]. However, cerebral IPC
dose not seem to be triggered by increased PGs, since indomethacin
failed to affect the protective effects of IPC. The negative role
for PGs pathway may be attributed to the specific model of focal
cerebral ischemia used for precondi-tioning. Saline infusion
compromised oxygen and glucose supply, however, inflammatory
response could be smaller, due to maintained idle perfusion at least
partial being able to prevent from disturbed microcirculation and
reduce leukocyte-endothelial cell adhesive interaction and so on.
This in turn potentially diminished the inflammatory signal
(including PGs pathway) as a trigger for preconditioning mechanisms.
The result suggested the delayed neuroprotection by our model of IPC
has different triggering mechanisms from that of delayed ischemic
tolerance induced by LPS.
Three types of COX isoforms have
been documented: COX-1, COX-2, and COX-3. COX-1 is constitutively
expressed under physiological conditions. COX-3 is identical in
sequence to COX-1 except for the in-frame retention of intron 1.
Like its counterpart COX-1, COX-3 does not generally appear to be
induced by acute inflammatory stimulation[14]. Several
studies have demonstrated that COX-2 played an important role in the
development of ischemic injury. In rodents as well as in humans,
cerebral ischemia up-regulated COX-2 in neurons, blood vessels, and
inflammatory cells in the injured brain[4,15,16].
Although COX-2 mRNA was not induced in the ischemic core, it was
induced in the penumbral area in permanent or transient MCAO in
rats. The up regulation of COX-2 began 6 h after ischemia, reached a
maximum at 12-24 h and subsided at 48 h[4,8,17]. NS398, a
selective COX-2 inhibitor, decreased infarction volume when
administered 6 h after induction of ischemia[18]. In
COX-2-deficient mice, there was a significant reduction in the MCAO-induced
brain injury[19,20]. Our results that COX-2 mRNA and
protein in the MCA cortex increased at 24 h after reperfusion in the
rats of MCAO and reperfusion group were consistent with these
previous studies. The mechanisms of the cytotoxicity of COX-2
induction may be related to the excessive release of arachidonic
acid, and its products such as PGs, thromboxane and free oxygen
radicals. These factors may act as the major inflammatory substances
involved in the pathogenesis of ischemia and reperfusion[8].
COX-2 reaction products may also contribute to NMDA-induced neuronal
injury and the pathogenesis of nitric oxide after ischemia[21,22].
Evidence has shown that COX-2 was an
obligatory effector in delayed IPC in the heart[13].
Increased expression of COX-2 protein was observed 24 h after IPC
with an increased enzymatic activity[23]. NS-398 and
celecoxib, two selective inhibitors of COX-2, abolished the
cardioprotection conferred by IPC [24]. It was also
demonstrated that the expression of COX-2 subsided 48 h after a
short period of ischemia in the brain[4,8,17]. Since the
delayed neuroprotection produced by IPC lasts as long as 7 d and is
much longer than that of delayed cardioprotection (72 h), the
up-regulated COX-2 does not seem to be the effector of delayed neuro-protection
by IPC in the brain. In the present study, we examined the
expression of COX-2 after IPC or IPC followed by ischemia and
reperfusion injury. Our results demonstrated that IPC had no direct
effect on the cortex COX-2 mRNA and protein expression 72 h later.
Thus, it is not likely that an up-regulation of COX-2 contributes to
the delayed neuroprotec-tion by IPC in the brain. In the present
study, we demonstrated that IPC significantly prevented the
up-regulation of COX-2 mRNA and protein expression with a reduced
infarction of the brain following ischemia and reperfusion injury.
These results are similar to the previous finding that hyperbaric
oxygen confers neuroprotection by depressing COX-2 expression and
activity following ischemia and reperfusion injury[8]. So
we speculate that while the up-regulation of COX-2 contributes to
the delayed cardioprotection by IPC in the heart, it is probably
that COX-2 takes an inverse role in delayed neuroprotection by IPC
in the brain. Our results are consistent with the recent observation
that COX-2 expression was down-regulated by ischemic preconditioning
in the gerbil brain[25].
Recently, it has been shown that
preconditioning stimulus reprogrammed the response of gene
transcription to subsequent ischemia and reperfusion injury in mice.
This feature mimics specific adaptive neuroprotective strategies
seen in hibernation[6]. Depressed protein synthesis may
be mediated in part via translational mechanisms that affect both
initiation and elongation[6,26]. Since IPC confers robust
neuroprotection, one presumption is that ischemic preconditioning
may induce pro-survival factors that would negatively regulate
COX-2. To date, three major mechanisms have been suggested to
contribute to the neuroprotection by IPC, including anti-excitotoxic
mechanisms, anti-apoptosis mechanisms, and anti-inflammatory
mechanisms[1]. COX-2 has been verified to take an
important role in pro-inflammatory action. We speculate that
down-regulation of COX-2 may cooperate with other protective
mechanisms and ultimately result in neuroprotection. However, more
studies are needed to verify this hypothesis.
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