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Introduction
Tetramethylpyrazine (TMPZ) is an active alkaloid
purified from a commonly used Chinese herb named "Chung
Chong" (Ligusticum wallichii Franchat), which has been
used for at least 2000 years by traditional Chinese
physicians to stimulate blood circulation, relieve pain (Figure
1)[1], and treat a variety of vascular diseases, notably ischemic
stroke and pulmonary hypertension secondary to chronic
obstructive pulmonary diseases[1,2]. It has been reported
that TMPZ improves changes in the microcirculation of patients with acute cerebral
thrombosis[3]. Ho et
al[4] showed that TMPZ increased the survival rate of Mongolian gerbils
with experimentally-induced stroke. Furthermore, TMPZ has
exhibited effective antiplatelet activity in both
in vitro and in vivo
studies[5,6].
Ischemic brain injury often causes irreversible brain
damage. The cascade of events leading to neuronal injury
and death in ischemia includes excitotoxicity, inflammation,
edema formation, apoptosis, and
necrosis[7]. As described previously, we demonstrated that TMPZ possesses marked
neuroprotective activity[8]. This preventive effect of TMPZ
on cerebral ischemic damage in middle cerebral artery
occlusion (MCAO)-reperfusion rats is assumed to be mediated, at
least partially, by the inhibition of platelet aggregation,
neutrophil activation, and inflammatory responses such as
inducible nitric oxide synthase (iNOS) expression, and
reactive oxygen species formation in cerebral ischemic areas.
Furthermore, several reports have also demonstrated that
TMPZ possesses neuroprotection against ischemic brain
injury through the reduction of neuronal apoptosis (eg, Bcl-xL)[9,10].
Recently, it was demonstrated that hypoxia-inducible
factor-1α (HIF-1α) expression increases in the rat brain
during cerebral ischemia induced by different models of arterial
occlusion or cardiac arrest[11]. The increased
HIF-1α protein level observed after ischemia-reperfusion was
presumably induced by the loss of the oxygen
supply[12], resulting in a greater extent of binding activity to the iNOS gene which
reaches a consequent peak of iNOS protein expression.
HIF-1α may bind to the iNOS promoter gene under hypoxic
condi-tions. Such binding is associated with an increase in iNOS
expression[13].
In our previous study[8], we partially resolved the
mechanisms of TMPZ in MCAO-induced transient focal cerebral
ischemia in rats; however, the detailed mechanisms
underlying the inhibitory effect of TMPZ in inflammatory and
apoptotic responses induced by MCAO have still not yet
been completely resolved. We therefore further examined
the effect of TMPZ on MCAO-induced cerebral ischemia,
and utilized the findings to further characterize the
neuroprotective effect of TMPZ.
Materials and methods
Animals Male Wistar rats (250_300 g) were used in this
study. All animal experiments and care were performed
according to the Guide for the Care and Use of Laboratory
Animals (National Academy Press, Washington DC, 1996).
Before undergoing the experimental procedures, all animals
were clinically normal and free of apparent infection or
inflammation, and showed no neurological deficits.
The animals were anesthetized with a mixture of 75% air and 25%
O2 gases containing 3% isoflurane. The rectal temperature was
maintained at 37±0.5 °C.
MCA occlusion assay The right MCA was occluded as
described previously[8,14]. Briefly, the right common carotid
artery was exposed and a 4-0 monofilament nylon thread (25
mm) coated with silicon was then inserted from the external
into the internal carotid artery until the tip occluded the
origin of the MCA. After closure of the operative sites, the
animals were allowed to awake from the anesthesia. During
another brief period of anesthesia, the filament was gently
removed after 1 h of MCAO.
Forelimb akinesia test An observer blinded to the
identity of the groups assessed the neurological deficits at 1 and
24 h after reperfusion (before sacrifice) using the forelimb
akinesia (also called the postural tail-hang) test, while the
spontaneous rotational test was used as a criterion for
evaluating the ischemic insult[15]. Animals not showing
behavioral deficits at the above time points after reperfusion were
excluded from the study. However, reperfusion was also
ensured by an improvement in the ipsilateral local blood flow
of at least 60% of the baseline following an initial sharp
decrease to about 30% of the baseline caused by MCAO as
determined using a continuous laser Doppler flowmeter
(Oxford Optronix, Oxford, UK) with a standard needle probe
(pp-051). The rats were sacrificed by decapitation after 24 h
of reperfusion. Their brains were cut into 2 mm coronal slices.
Each stained brain (2% 2,3,5-triphenyltetrazolium, TTC) slice
was drawn using a computerized image analyzer (Image-Pro
Plus, Media Cybernetics, Silver Spring, MD, USA). The
calculated infarct areas were then compiled to obtain the infarct
volumes (mm3) for each brain. Infarct volumes were expressed
as a percentage of the contralateral hemisphere volume
using the formula [the area of the intact contralateral (left)
hemisphere _ the area of the intact region of the ipsilateral (right)
hemisphere] to compensate for edema formation in the
ipsilateral hemisphere[14]. All of the animals were divided into 3
groups: (i) a sham-operated group; (ii) a solvent
solution-treated group (cremophor: ethanol: normal saline, 1:1:4); and
(iii) a single dose (20 mg/kg, ip) of TMPZ (Aldrich, Milwaukee,
MI, USA)-treated group. In the group treated with the
solvent or TMPZ, the rats were given isovolumetric solvent or
TMPZ (20 mg/kg) 20 min before MCAO.
Preparation of brain tissue sample The MCAO-insulted
and sham-operated rats were anesthetized with chloral
hydrate (400 mg/kg, ip), then the apex of the heart was
penetrated with a perfusion cannula inserted through the left
ventricle into the ascending aorta. Perfusion with ice-cold
phosphate-buffered saline was performed, and an incision
was made in the right atrium for venous drainage. Fresh
brains were removed and sectioned coronally into 4
sequential parts from the frontal lobe to the occipital lobe. The third
part (3_9 mm from the frontal lobe) of each hemisphere was
separately collected, snap-frozen in liquid nitrogen, and
stored at -70 °C.
Western blot assay Expression of HIF-1α and activated
caspase-3 in the brain were analyzed by Western blotting as
described by Rodrigo et al
[16] with some modifications. The
frozen tissues were weighed and placed in ice-hold
homogenate buffer at a ratio of 1 g tissue to 1 mL buffer. Each brain
tissue sample was homogenized using a polytron
homo-genizer, then sonicated for 10 s 3 times at 4 °C. The
sonicates were subjected to centrifugation (at 10
000×g).The supernatant (50 µg protein) was subjected to SDS-PAGE and
electrophoretically transferred to PVDF membranes (0.45 µm;
Hybond-P, Amersham, Buckinghamshire, HP, UK). After
incubation in blocking buffer (10 mmol/L Tris-base, 100
mmol/L NaCl, 0.1% Tween 20, and 5% dry-skim milk, pH 7.5)
and after washing 3 times with TBST buffer (10 mmol/L
Tris-base, 100 mmol/L NaCl, and 0.1% Tween 20, pH 7.5), the
blots were hybridized with an anti-HIF-1α polyclonal
antibody (1:1000, R&D, Minneapolis, CA, USA), and an
anti-active caspase-3 pAb (1:250, BioVision, Mountain View, CA,
USA) or an anti-actin mAb (1:7000, Sigma, St Louis, MO,
USA) in TBST buffer overnight. Blots were subsequently
washed 4 times with TBST and incubated with secondary
horseradish peroxidase-conjugated goat anti-mouse mAb
(Amersham) for 1 h. The blots were then washed and the
immunoreactive protein was detected using film exposure
with enhanced chemiluminescence detection reagents
(Amersham).
Isolation of total RNA and RT-PCR Total RNA was
isolated from the ipsilateral cortex by a commercially available
kit (TRIzol, Gibco, Grand Island, NY, USA) according to the
manufacturer's instructions. For each RT-PCR reaction, 0.5
mg of the RNA sample and 0.2 µmol/L of primers were
reverse-transcribed and amplified in a 50 µL of reaction
mixture of commercially available reagents (SUPERSCRIPT
One-Step RT-PCR with PLATINUM Taq Kit, Invitrogen, Carlsbad,
CA, USA) containing a 1× reaction mixture and 0.2 µmol/L of
an RT/Taq mixture in 1 cycle of 30 min at 50 °C for reverse
transcription and 1 cycle at 95 °C for 3 min; followed by 40
cycles at 95, 62, and 72 °C for 30, 40, and 40 s, respectively;
with a single extension step at 72 °C for 5 min, followed by
4 °C for amplification in a thermal cycler (GeneAmp PCR
system 2400, Perkin-Elmer, Wellesley, MA, USA). For
visualization and quantification by densitometry of each RT-PCR
reaction, a 10 µL aliquot was subjected to electrophoresis on
a 1.5% agarose gel using a mini horizontal submarine unit
(HE 33) containing 0.5 mg/mL ethidium bromide to allow
UV-induced fluorescence (TCP-20.M, Vilber Lourmat, Marne-
la-Vallee Cedex, France).
Anti-oxidative activity in rat brain homogenate
preparations Rat brain homogenates were prepared from the brains
of freshly killed Wistar rats, and the peroxidation in the
presence of iron ions was measured by the thiobarbituric acid
method, as described by Braughler et
al[17], with some modi-fications. In brief, whole brain tissue, excluding the
cere-bellum, was washed and homogenized in 10 volumes of
ice-cold Krebs buffer using a homogenizer (Glas-col, Millville,
NJ, USA). The homogenate was centrifuged at low speed
(1000×g) for 10 min, and the resulting supernatant (adjusted
to 2 g/L) was used immediately in the lipid peroxidation assay.
The reaction mixture with TMPZ or vehicle solution (0.5%
DMSO) was incubated for 10 min, then stimulated by the
addition of a ferrous ion (200 mmol/L, freshly prepared), and
maintained at 37 °C for 30 min. The reaction was terminated
by adding 10 µL of ice-cold trichloroacetic acid solution [4%
(w/v) in 0.3 mol/L HCl] and 200 µL of thiobarbituric
acid-reactive substance reagent [TBARS, 0.5%
(w/v) thiobar-bituric acid in 50%
(v/v) acetic acid]. After boiling for 15 min,
the samples were cooled and extracted with
n-1-butanol. The extent of lipid peroxidation was estimated by TBARS and
was read at 532 nm in a spectrophotometer (Model U3200,
Hitachi, Tokyo, Japan). Tetramethoxypropane was used as a
standard, and the results were expressed as nanomoles of
malondialdehyde equivalents per milligram protein of the
supernatant of rat brain homogenates. The protein contents
of the brain homogenates and other preparations were
determined with the Bio-Rad method.
Statistical analysis The experimental results are
expressed as the mean±SEM and are accompanied by the
number of observations. Student's unpaired
t-test was used to determine significant differences in the study of
MCAO-induced cerebral ischemia. The other experiments were
assessed by ANOVA. If this analysis indicated significant
differences compared with the group means, then each group
was compared using the Newman-Keuls method. A
P value of less than 0.05 was considered statistically significant.
Results
Treatment with TMPZ can reduce the infarct area in rat
brain The animals of all the groups in this study showed
similar physiological values for rectal temperature, mean
arterial blood pressure, plasma glucose, and hematocrit (%)
before, during, and after MCAO (data not shown). Neither
abnormal behavior, depression of respiratory function, nor
hypothermia was observed in the solvent- or TMPZ-treated
groups. Cerebral infarction was examined in 2 mm-thick slices
of the cerebrum of MCAO-reperfused rats through TTC
staining. In our previous report[8], administration of TMPZ
at 10 and 20 mg/kg produced concentration-dependent
reductions in infarct volumes compared with the solvent group.
In this study, we further showed the statistical results of the
infarct areas of the solvent- and TMPZ (20 mg/kg)-treated
groups at various distances from the frontal pole (Figure 2).
Treatment with TMPZ (20 mg/kg) reduced the infarct area in
all regions, especially in the third to fifth sections (Figure 2).
In the solvent-treated rats, approximately 58.6%±4.1% of the
entire area was infarcted in the third section, while TMPZ (20
mg/kg) ingestion reduced the area to 37.6%±4.7% in the third
section (Figure 2).
TMPZ treatment can reduce expression of
HIF-1α and activated caspase-3 in the ischemic rat brain
We further examined the expression of HIF-1α when treated with TMPZ
in the ischemic rat brain. As shown in Figures 3 and 4, the
expression of HIF-1α and activated caspase-3 were
upregu-lated after MCAO-reperfusion injury. TMPZ (20 mg/kg)
treatment significantly (P<0.05) suppressed the level of
HIF-1α and activated caspase-3 in the ipsilateral hemisphere (Figures
3 and 4).
TMPZ treatment can reduce the transcription of
TNF-α gene in the ischemic rat brain Transient MCAO also
resulted in a significant and more sustained increase in the
expression of TNF-α mRNA in the injured hemisphere
compared with the levels obtained in the corresponding areas of
the sham-operated group (Figure 5A). TMPZ (20 mg/kg)
treatment significantly reduced this reaction (Figure 5A).
In addition, TMPZ was further tested for its ability to
inhibit non-enzymatic lipid peroxidation in rat brain
homogenates stimulated by a ferrous ion. At 0.5_5 mmol/L,
TMPZ did not significantly inhibit the ferrous-induced lipid
peroxidation in normal (Figure 5B) or MCAO-insulted rat
brain homogenates (n=3, data not shown). In addition, TMPZ
(0.5_5 mmol/L) also did not inhibit the ferrous-induced lipid
peroxidation in rat brain homogenates. TMPZ did not
interfere with the thiobarbituric acid test, since the color
formation was unchanged if it was added after the incubation with
thiobarbituric acid reagents. However, α-tocopherol (200
µmol/L) inhibited ion-dependent lipid peroxidation in this
reaction (data not shown).
Discussion
TMPZ can permeate the blood_brain barrier and can be
enriched in the brain, especially the brainstem. The present
study demonstrates that MCAO-reperfusion injury induces
increases in HIF-1α and active caspase-3 protein expressions,
and TNF-α mRNA expression, which may represent the
response of neurons suffering from the ischemic insult. In
fact, the increase in HIF-1α expression after an ischemic
insult may exert opposite effects on neuronal
fates[11, 12]. HIF-1α activation of target genes related to vascularization,
glucose transport, and glycolytic metabolism may be regarded
as an adaptive response to ischemic conditions that may
promote neuronal survival within ischemic areas. However,
in other instances, the increased activation of
HIF-1α may act as a noxious signal for neuronal survival.
HIF-1α, which combines with p53, may promote apoptotic cell death in
ischemic areas[11].
In this study, we showed that the elevation of active
caspase-3 expression occurred in the same time frame as
HIF-1α expression after ischemic injury, and these
expressions could be significantly suppressed by pretreatment with
TMPZ (20 mg/kg).
It has been reported that several apoptosis-related genes,
including caspase-9 and -3, are all strongly expressed after
ischemic injury[18]. In addition, hypoxia may cause
HIF-1α to bind to p53 in order to stabilize it, and also activates the
expression of various genes, including bax (a pro-apoptotic
member of Bcl-2 family proteins)[18]. Bax is translocated to
the mitochondria where it releases cytochrome c into the
cytosol to interact with Apaf-1 to activate caspase-9, which
in turn activates downstream caspases, such as
caspase-3[19].
TNF-α is one of the key immunomodulatory and
pro-inflammatory cytokines upregulated during brain
ischemia[20]. Administration of TNF-α during an ischemic brain insult has
been shown to augment the injury, as evidenced by increased
tissue damage and neurological
deficits[20]. In addition to inflammation,
TNF-α has also been shown to be involved in
apoptosis[21]. In our study, we also found TMPZ can
downregulate the transcription of TNF-α during brain
ischemia. Therefore, TMPZ inhibition of active caspase-3
expression may occur, at least partially, through the
inhibition of TNF-α expression in ischemic brain injury.
The phospholipid bilayers of cellular and subcellular
membranes are undoubtedly major targets for free radicals.
The compound that inhibits membrane phospholipid peroxidation seems to exert a pharmacological effect in the
prevention of radical-induced oxidative pathological events.
Among cell-free systems, brain homogenates are usually
chosen to evaluate an anti-oxidant's effects on lipid
peroxidation[17]. Rat brain homogenates exposed to a
ferrous ion exhibit lipid peroxidation in air by a mechanism whose
induction step may primarily involve site-bound,
iron-mediated decomposition of lipid hydroperoxides to yield alkoxy
or peroxyl radicals, leading to the chain reaction of lipid
peroxidation[22]. In this system, TMPZ did not effectively
inhibit lipid peroxidation, indicating that the inhibition of
lipid peroxidation might not be the neuroprotective
mechanism for TMPZ in ischemic brain injury.
In conclusion, we found that the neuroprotective effect
of TMPZ on cerebral ischemic damage in MCAO-reperfusion
rats is probably mediated by the inhibition of
HIF-1α and TNF-α activation, followed by the inhibition of apoptosis
(active caspase-3). The rationale for the use of TMPZ is
based on the fact that the multiple deleterious process in
different cell types of organelles are initiated during
ischemia-reperfusion injury which ultimately synergistically moves
toward irreversible injury. Therefore, treatment using TMPZ
is not limited to 1 factor, but involves many mechanisms,
most of which may be interrelated. We speculate that the
correction of these molecules and morphological changes
may lead to neurobehavioral improvement in patients; thus,
treatment using TMPZ may represent an ideal approach for
improving function after ischemia-reperfusion brain injury.
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