Activation of Peroxisome Proliferatoractivated Receptor b/d Attenuates Acute Ischemic Stroke on Middle Cerebral

Ischemia Occlusion in Rats

Xiaodong Chao, PhD,*1 Chunlei Xiong, MD,1 Weifeng Dong, MD,x1 Yan Qu, PhD,jjWeidong Ning, MD,{ Wei Liu, PhD,jj Feng Han, MD,jj Yijie Ma, MD,#

Fe

Stroke is the second leading cause of death in industri-

alized countries and the most important cause of acquired

a situation associated with increased mortality and

long-term functional disability.2 The mechanisms under-

Department of Cardiology, Tangdu Hospital, Fourth Military Medi-

cal University, Xian; xDepartment of Cardiology, The First HospitalFoundation (2012M512099).

Address correspondence to Hua Han, Department of Medical Ge-Corps Hospital, Urumqi, China.of PLA, Lanzhou; jjDepartment of Neurosurgery, Xijing Institute ofClinical Neuroscience, Xijing Hospital, Fourth Military Medical Uni-

versity, Xian; {Department of Neurosurgery, Peoples Hospital ofJishan County, Shanxi; and #Department of Neurosurgery, Xinjiang

Received September 22, 2013; revision received November 7, 2013;

netics and Developmental Biology, Fourth Military Medical Univer-

sity, Xian 710038, China. E-mail: kelyue@msn.com.1These authors contributed equally to this work.

1052-3057/$ - see front matter

2013 by National Stroke Associationhttp://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2013.11.021adult disability.1 Nearly one third of patients with acutelying neural cell injury in cerebral ischemic are not yet

From the *Department of Medical Genetics and Developmental

Biology, Fourth Military Medical University, Xian; Department of

Neurosurgery, Urumqi General Hospital of PLA, Urumqi;

X.C., C.X., and W.D. contributed equally to this work.

This work was financially supported by the National Natural Sci-

ence Foundation of China (81301075) and China Postdoctoral ScienceIntroduction ischemic stroke develop early neurologic deterioration,accepted November 23, 2

Journal of Stroke and Ction factor that belongs to the nuclear hormone receptor family. There is little infor-

mation about the effects of the immediate administration of specific ligands of

PPAR-b/d (GW0742) in animal models of acute ischemic stroke. Using a rat model

of middle cerebral ischemia occlusion (MCAO) in vivo, we have investigated the ef-

fect of pretreatment with GW0742 before MCAO.Methods: The neuroprotective ef-fect of GW0742 against acute ischemic strokewas evaluated by the neurologic deficit

score (NDS), drywet weight, and 2,3,5-triphenyltetrazolium chloride staining. The

levels of interleukin (IL)-1b, nuclear factor (NF)-kB, and tumor necrosis factor

(TNF)-awere detected by an enzyme-linked immunosorbent assay. The expressions

of inducible nitric oxide synthase (iNOS), Bax, and Bcl-2 were detected by Western

blot. The apoptotic cells were counted by in situ terminal deoxyribonucleotidyl

transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assay.

Results: The pretreatment with GW0742 significantly increased the expression ofBcl-2, and significantly decreased in the volume of infarction, NDS, edema, expres-

sions of IL-1b, NF-kB, TNFa, and Bax, contents of iNOS and the apoptotic cells in

infarct cerebral hemisphere comparedwith rats in the vehicle group at 24 hours after

MCAO. Conclusions: The study suggests the neuroprotective effect of the PPAR-b/d ligand GW0742 in acute ischemic stroke by a mechanism that may involve its anti-

inflammatory and antiapoptotic action. Key Words: PPAR-b/dGW0742middle

cerebral artery occlusion (MCAO)anti-inflammatoryrat.

2013 by National Stroke AssociationBackground: Peroxisome proliferatoractivated receptor (PPAR)-b/d is a transcrip-Rencong Wang, MD,jj Zhou013.

erebrovascular Diseases, Vol. -, No. - (---i, PhD,jj and Hua Han, PhD*), 2013: pp 1-7 1

well understood, but many studies have clearly shown MCAO and at reperfusion. The right femoral artery was

groups consisting of 8 animals. The first subgroup was

analyzed by the image analyzing system (Adobe Systems

X. CHAO ET AL.2Materials and Methods

Animals

Adult male SpragueDawley rats weighing 260-280 g

and aged 11-12 weeks were purchased from Xian Jiaotong

University Animal Services for this study. The protocol

was approved by the institutional animal care and use

committee and the local experimental ethics committee.

All rats were allowed free access to food and water before

surgery under optimal conditions (12/12 hours light/dark

with humidity of 60 6 5% and temperature of 22 6 3C).

Model of MCAO

MCA thread occlusion was induced according to the

previously published methods.15 Rats were anesthetized

with chloral hydrate at the dose of 380 mg/kg, intraperi-

toneally. The right common carotid artery was exposed

and separated carefully from the vagus nerve and ligated

at the more proximal side through a right paramedian

incision. The external carotid artery was ligated. The oc-

cipital and pterygopalatine arteries were coagulated.

Ischemia was produced by advancing a tip rounded .30-

mm nylon suture into the internal carotid artery through

the external carotid artery. Reperfusion was produced by

withdrawal of the intraluminal nylon suture. The physio-

logical variables in rats weremeasured at baseline, duringPPAR-b/d exerts a strong protection from renal and gut

ischemiareperfusion (I/R) injury.8,9 And many studies

had clearly shown that GW0742, a potent PPAR-b/

d agonist, exerts anti-inflammatory effects in atheroscle-

rosis, myocardial infarction, endotoxic shock, and lung

inflammation.10-13 Recent research indicated that PPAR-

b/d plays an important role in integrating and regulating

central inflammation and food intake after middle

cerebral ischemia occlusion (MCAO).14

So the aim of our present studywas to examine the neu-

roprotective effect of the PPAR-b/d ligand GW0742 on

MCAO in rats and its potential mechanisms.that inflammatory response and oxidative stress have

been found to play an important role in the pathogenesis

of cerebral ischemia.3

Peroxisome proliferatoractivated receptors (PPARs)

are ligand-activated transcription factors that belong to

the nuclear hormone receptor superfamily.4 There are

three members of the PPAR subfamily: PPAR-a, PPAR-

b/d, and PPAR-g. PPARs have been implicated in the

pathogenesis of a number of diseases, including diabetes

mellitus, obesity, atherosclerosis, and neurologic dis-

eases.5 There is good evidence that WY14643, a selective

PPAR-a agonist, and rosiglitazone and pioglitazone, two

PPAR-g agonists, have the neuroprotective effect on cere-

bral ischemia.6,7 However, there is less information about

the role of ligands of PPAR-b/d in acute ischemic stroke.Incorporated, San Jose, CA). To compensate for the effect

of brain edema, the corrected volume was calculated

using the following equation: percentage hemisphere

lesion volume (% HLV) 5 {[total infarct volume 2 (right

hemisphere volume 2 left hemisphere volume)]/leftphate-biotin nick end labeling (TUNEL). The sham

groupwas only subjected to surgical procedures, whereas

other animals were subjected to focal ischemia by MCAO

using intraluminal thread, and after 2 hours of MCAO re-

perfusion allowed by retracting the thread.

Evaluation of Neurologic Impairment

Twenty-four hours after MCAO, the neurologic deficit

of each rat was evaluated according to the Longa method

of a 5-point scale16 by the same experimenter, who was

blinded to the different treatments in the experiment

(no neurologic deficit 5 0, failure to extend right paw

fully5 1, circling to right5 2, falling to right5 3, and be-

ing unable to walk spontaneously and depression of

consciousness 5 4).

Measurement of Infarct Volume

After the NDS, rats were killed and their brains quickly

removed and frozen at 220C for 15 minutes. Coronalbrain sections (2-mm thick) were stained with 2% 2,3,5-

triphenyltetrazolium chloride at 37C for 20 minutes fol-lowed by fixation with 4% paraformaldehyde. Brain slices

were scanned individually and the unstained area wasused for neurologic deficit score (NDS) and 2,3,5-

triphenyltetrazolium chloride staining; the second sub-

group was used for brain water content; the third

subgroup was used for detecting nuclear factor (NF)-kB,

tumor necrosis factor (TNF)-a, and interleukin (IL)-1b

by an enzyme-linked immunosorbent assay; the fourth

subgroup was used for detecting inducible nitric oxide

synthase (iNOS), Bcl-2, and Bax by Western Blot; and

the fifth subgroup was used for terminal deoxyribonu-

cleotidyl transferase-mediated deoxyuridine triphos-cannulated for continuous monitoring of blood pressure

and arterial blood sampling. A rectal probe was inserted

to monitor core temperature. Arterial blood gases and

plasma glucose were measured at baseline, during

MCAO and at reperfusion.

Experimental Protocol

Male SpragueDawley rats were randomly divided

into 3 groups: sham group; vehicle group, which was pre-

treated with saline at 30 minutes before MCAO; GW0742

group, which was pretreated with GW0742 .3 mg/kg

(intraperitoneally) at 30 minutes before MCAO. The

rats in each of the groups were subdivided into 5 sub-

hemisphere volume} 3 100%. Infarct volume measure-

nutes at 4 C to separate the sample into supernatant A and

4C. The primary antibodies and their dilutions were as

UK; 1:5000). After being extensively rinsed with Tris-buff-

Q1ACTIVATION OF PPAR ATTENUATES ACUTE ISCHEMIC STROKE 3follows: Bcl-2 andBax (SantaCruz Inc, 1:200); iNOS (Signal

Transduction, 1:1000); and b-actin (Abcam Inc, London,pellet A. Supernatant A, containing the cytosolic/mito-

chondrial protein, was further centrifuged at 12,000g for

30 minutes at 4C to separate supernatant B from pelletB. Supernatant Bwasused as the cytosolic fraction andpel-

let Bwasusedas themitochondrial fraction after resuspen-

sion in buffer. The protein samples were separated on 10%

or 12% sodium dodecyl sulfatepolyacrylamide gel elec-

trophoresis gels (30 mg/lane) and then transferred to a

nitrocellulose membrane (Bio-Rad, Hercules, CA). The

membrane was blocked with 5% nonfat dry milk in Tris-

buffered saline containing 0.1% Triton 3-100 buffer and

then incubated with primary antibodies overnight atSamples were collected from the ischemic brain tissue.

Whole-cell lysates were obtained by homogenizing the

samples with a homogenizer in 5 volumes of buffer

(20 mmol/L of 4-(2hydroxyethyl)-1-piperazineethanesul-

fonic acid, 1.5 mmol/L of MgCl2, 10 mmol/L of KCl,

1 mmol/L of ethylenediaminetetraacetic acid, 1 mmol/L

of ethylene glycol tetraacetic acid, 250 mmol/L of sucrose,

.1 mmol/L of phenylmethyl sulfonyl fluoride, 1 mmol/L

of dithiothreitol, and proteinase inhibitor cocktail tablets;

pH7.9). Sampleswere furthercentrifugedat 750g for 15mi-ments were carried out by an investigator blinded to the

treatment groups.

Evaluation of Cerebral Edema

Twenty-four hours after MCAO, cerebral edema was

determined by measuring the brain water content accord-

ing to the wetdry method.17 The rats were killed with

chloral hydrate anesthesia, and the brain was rapidly

removed and dissected. Brain samples from the ischemic

hemisphere were immediately weighed on an electronic

balance and then dried in an oven at 110C for 24 hoursto obtain the dry weight. Brain water content was calcu-

lated as follows: brain water content (%) 5 (wet

weight 2 dry weight) 3 100/wet weight.

Enzyme-linked Immunosorbent Assay

Twenty-four hours after MCAO, rats were sacrificed and

thebrain tissuehomogenateswere obtained from the infarct

cerebral hemisphere. The contents ofNF-kB, TNF-a, and IL-

1b were measured in brain tissue homogenates using spe-

cific enzyme-linked immunosorbent assay kits according

to the manufacturers instructions (NF-kB: Themicon Inter-

national Inc Temecula, CA; TNF-a: Uscn Life, Wuhan,

China; IL-1b: Bender Medsystems, Burlingame, CA).

Western Blot

Brains were quickly removed at 24 hours after MCAO.ered saline containing 0.1% Triton3-100 buffer, the mem-

branes were incubated with secondary antibodies (Santa

Cruz Inc, Santa Cruz, CA; 1:2000) for 1 hour at room tem-

perature, and then the bar graph depicts the ratios of semi-

quantitative results obtained by scanning reactive bands

and quantifying the optical density using Videodensitom-

etry (Bio-1D vers. 99 image software, Bio-Rad).

TUNEL Assay

ATUNEL assay was used to assess DNA damage. The

sections were treated as instructed with an in situ cell

death detection kit (Roche, Basel, Switzerland). Diamino-

benzidine (Sigma St. Louis, MO) was used as a chro-

mogen. TUNEL-positive cells displayed brown staining

within the nucleus of apoptotic cells. DNA fragmentation

was quantified under high-power magnification (3400)

by an investigator who was blinded to the studies and

was expressed as number per square millimeter.

Statistical Analysis

Data are expressed as the means 6 standard deviation.

Statistical evaluation of the data was performed by an

analysis of variance. All assays were conducted in tripli-

cate. Avalue of P less than .05 was considered statistically

significant.

Results

Physiological Parameters

There were no significant differences for these variables

(mean arterial blood pressure, arterial carbon dioxide par-

tial pressure, arterial oxygen partial pressure, pH, rectal

temperature, and blood glucose) at baseline, during

MCAO and at reperfusion. These variables remained in

the normal range during the experimental period

observed (Table 1).

Effects of Pretreatment with GW0742 on Neurologic

Deficit and Infarction

To determine the neuroprotective effect of GW0742

against cerebral ischemic injury, we examined the neuro-

logic deficit and infarct volume at 24 hours after MCAO.

No infarct and neurologic deficit was observed in the

sham group rats, in contrast, cerebral infarct volume

and NDSs in the vehicle group rats were significantly

higher (Fig 1A-C). As shown in Figure 1A-C, pretreat-

ment with GW0742 decreased cerebral infarct volume

and ameliorated neurologic deficit induced by MCAO.

Effect of Pretreatment with GW0742 on Brain Edema

Formation

As shown in Figure 1D, the brain water content in the

ischemic area of the vehicle group rats was significantly

higher than that of the sham group rats at 24 hours after

MCAO. Pretreatment with GW0742 significantly reduced

brain edema in comparison with the vehicle group rats.

Effect of Pretreatment with GW0742 on the Expressions

of NF-kB, TNF-a, and IL-1b

To investigate the anti-inflammatory effect of GW0742

on MCAO rats, we examined expressions of NF-kB,

TNF-a, and IL-1b in infarct cerebral hemisphere at

24 hours after MCAO. Table 2 shows that the expression

contents of NF-kB, TNF-a, and IL-1b in the vehicle group

were much higher than that in the sham-operated group.

Pretreatment with GW0742 significantly also brought

down increased expression contents of NF-kB, TNF-a,

Effect of Pretreatment with GW0742 on Expression of

iNOS

As shown in Figure 2, the expression of iNOS signifi-

cantly increased in the vehicle group compared with the

sham group. Pretreatment with GW0742 significantly

brought down the increased expression of iNOS at

24 hours after MCAO.

Effect of Pretreatment with GW0742 on Expressions of

Bax and Bcl-2

To investigate antiapoptotic action of GW0742 on

MCAO rats, we examined expressions of Bax and Bcl-2

in infarct cerebral hemisphere at 24 hours after MCAO.

at 24 hours after MCAO in rats. (A) Represen-

tative coronal brain sections (2-mm thick) from

Table 1. Physiological parameters

Time interval T (C)

Arterial blood gases

MAP (kPa) PaCO2 (kPa) PaO2 (kPa) pH Glucose (mM)

Preischemia 37.4 6 .44 14.5 6 1.2 6.4 6 .9 32.3 6 5.8 7.36 6 .08 4.78 6 .58Intraischemia 37.3 6 .46 14.3 6 1.7 6.6 6 .5 31.8 6 6.5 7.34 6 .06 4.69 6 .53Reperfusion 37.4 6 .37 14.4 6 1.1 6.5 6 1.1 30.9 6 6.9 7.35 6 .07 4.57 6 .62

Abbreviations: MAP, mean arterial pressure; PaCO2, arterial carbon dioxide partial pressure; PaO2, arterial oxygen partial pressure; T, rectaltemperature.

Variables are presented as means6 standard deviation. All values were within normal range, and there were no significant differences amonggroups (n 5 8 each).

X. CHAO ET AL.4and IL-1b of rats at 24 hours after MCAO.GW0742-pretreated rats stained with 2% TTC

showing infarction. Red colored regions in the

TTC-stained sections indicate nonischemia

and pale colored regions indicate ischemia

portion of the brain. (B) The histogram shows

the total infarct volume of rats. Mean 6 SD.

*P , .01 versus the sham group; #P , .05

versus the vehicle group, n 5 8 each. (C) The

histogram shows the neurologic deficit scoresAs shown in Figure 3, expressions of Bax and Bcl-2

Figure 1. Effect of GW0742 on infarction,

neurologic deficit, and brain edema formationof rats. Mean 6 SD. *P , .01 versus the

sham group; #P, .05 versus the vehicle group,

n5 8 each. (D) The histogram shows the brain

water content of rats. Mean 6 SD. *P , .01

versus the sham group; #P , .05 versus the

vehicle group, n 5 8 each. Abbreviations:

MCAO, middle cerebral ischemia occlusion;

SD, standard deviation; TTC, 2,3,5-

triphenyltetrazolium chloride. For interpreta-

tion of the references to color in this figure

legend, the reader is referred to the web version

of this article.

PPAR-b/d plays an important role in acute ischemic

stroke. The mechanisms underlying the biological effects

induced by activation of PPAR-b/d are mediated through

a number of potential pathways. Ding et al21 found that

ligands of PPAR-b/d can lead to anti-inflammatory activ-

ities and interfere with NF-kB signaling. It is known from

the literature that NF-kB is activated in tissue ischemia

and reperfusion, which is a protein that acts as a switch

to turn inflammation on and off in the body. We also

found that NF-kB was significantly increased in rat at

24 hours after MCAO, which can exacerbate cerebral

injury induced by I/R. In the present study, we found

that pretreatment with GW0742 can significantly reduce

I/R-induced activation of cerebral NF-kB after reperfu-

Table 2. Effect of GW0742 on the cerebral contents of NF-kB, TNF-a, and IL-1b at 24 hours after middle cerebral ischemia

me-linked immunosorbent assay

TNF-a (pg/mg protein) IL-1b (pg/mg protein)

2.13 6 .24 9.35 6 .423.85 6 .39* 12.91 6 .82*2.84 6 .27z 10.84 6 .61z

NF-a, tumor necrosis factor a.

ACTIVATION OF PPAR ATTENUATES ACUTE ISCHEMIC STROKE 5were significantly increased and decreased, respectively,

in the vehicle group compared with the sham group.

And pretreatment with GW0742 reduced Bax expression,

whereas increased Bcl-2 expression.

Effect of Pretreatment with GW0742 on in Situ Labeling

of DNA Fragmentation

Cerebral apoptosis was determined by TUNEL

staining. In the TUNEL assay, a large number of

TUNEL-positive cells were observed in infarct cerebral

hemisphere of rats, whereas TUNEL-positive cells were

not detected in the sham group rats. The number of

TUNEL-positive cells was significantly reduced in the

pretreatment with the GW0742 group compared with

the vehicle group at 24 hours after MCAO (Fig 4).

Discussion

In the present study, we first investigated the neuropro-

tective effect of GW0742 in a model for MCAO. Acute

ischemic stroke is a major cause of disability and death

in developed countries, and because of limited therapeu-

tic strategies there is increasing interest in prophylactic

pharmacologic treatment.18 Inoue et al19 found resvera-

trol, a dual agonist for PPAR-a and PPAR-g, protects the

murine brain from stroke in a PPAR-adependent

manner. After that more studies found that PPAR-a ago-

nists and PPAR-g ligands have neuroprotective effects

on brain ischemic injury. Recently, GW0742, specific and

occlusion in rats by an enzy

Group NF-kB (mg/mg protein)

Sham 6.08 6 .69Vehicle 17.54 6 1.43*GW0742 13.19 6 1.27y

Abbreviations: IL-1b, interleukin 1b; NF-kB, nuclear factor-kB; TAll values are expressed as means 6 standard deviation.*P , .001 versus the sham group.yP , .01 versus the vehicle group.zP , .05 versus the vehicle group (n 5 8 each).potent PPAR-b/d agonist, exerts beneficial effects in ani-

mal models of I/R injury.5,20 In the present study, we

found that pretreatment with GW0742 significantly

reduced the infarct volumes, the NDSs, and cerebral

edema of rats submitted to MCAO compared with rats

in the vehicle group at 24 hours after MCAO. These

results suggest the neuroprotective effect of GW0742 in

MCAO model of rats.

The mechanisms underlying neuronal injury in acute

ischemic stroke are not yet well understood. However,

accumulating evidence demonstrated that postischemic

inflammation contributes to the development of neuronal

injury and cerebral infarction. It has been well known thatsion. NF-kB plays a central role in the regulation ofFigure 2. Effect of GW0742 on the expression of iNOS at 24 hours after

MCAO in rats. Brain tissue homogenates were obtained from the injured ce-

rebral hemisphere and the expression of iNOSwasmeasured byWestern blot.

Data were normalized with b-actin. Data were expressed as the mean6 SD.

*P , .01 versus the sham group; #P , .05 versus the vehicle group, n 5 8

each. Abbreviations: iNOS, inducible nitric oxide synthase; MCAO, middle

cerebral ischemia occlusion; SD, standard deviation.

X. CHAO ET AL.6many genes responsible for the generation of mediators

or proteins in inflammation. These include the genes for

TNF-a, IL-1b, and iNOS.22 There is good evidence that

TNF-a and IL-1b, as proinflammatory cytokines,

contribute to pathogenesis, exacerbating I/R brain tissue

damage.23 We also found that TNF-a and IL-1b were

significantly increased in rat at 24 hours after MCAO.

Various studies have clearly reported that inhibition of

TNF-a formation significantly prevents the development

of the inflammatory process.24 Di et al found that

GW0742 can attenuate TNF-a and IL-1b production in

acute lung injury. In the present study, we found that

pretreatment with GW0742 can significantly reduce I/R-

induced production of cerebral TNF-a and IL-1b after re-

perfusion. It is clear that activation of TNF-a and IL-1b

can increase iNOS, cyclo-oxygenase-2, and various che-

mokine productions, which can raise brain tissue inflam-

mation and destruction. High levels of NO production

generated by iNOS are an important mediator associated

with the cerebral damage during I/R injury. In the present

study, we found that the expression of iNOS was signifi-

cantly increased in rat at 24 hours after MCAO and pre-

treatment with GW0742 can significantly bring down

the increased expression of iNOS. Therefore, the inhibi-

Figure 3. Effect of GW0742 on the expressions of Bcl-2 and Bax at 24 hours

after MCAO in rats. Brain tissue homogenates were obtained from the

injured cerebral hemisphere and the expressions of Bcl-2 and Bax were

measured by Western blot. Data were normalized with b-actin. Data were

expressed as the mean 6 SD. *P , .01 versus the sham group; #P , .05

versus the vehicle group, n5 8 each. Abbreviations: MCAO, middle cerebral

ischemia occlusion; SD, standard deviation.tion of the production of TNF-a, IL-1b, and iNOS by

GW0742 described in the present study is most likely25

Figure 4. Effect of GW0742 on DNA fragmentation at 24 hours after

MCAO in rats. (A) Representative photomicrographs of TUNEL staining

in the MCA territory of the ischemic cortex (3400). The arrows represent

TUNEL positive cells. (B) Quantitative analysis showed that GW0742 pre-

treatment reduced the number of TUNEL-positive cells in theMCA territory

of the ischemic cortex. Results are expressed as the mean 6 SD. *P , .001

versus the sham group; #P , .05 versus the vehicle group, n 5 8 each. Ab-

breviations: MCAO, middle cerebral ischemia occlusion; SD, standard devi-

ation; TUNEL, terminal deoxyribonucleotidyl transferase-mediated

deoxyuridine triphosphate-biotin nick end labeling.attributed to the inhibitory effect of activated NF-kB.

It is now well known that apoptosis during I/R injury

plays a major role in brain injury associated with stroke.26

Recently, it has been demonstrated that the PPAR-b/d ago-

nists possess potent antiapoptotic properties in vitro.27

Bax, a proapoptotic gene, plays an important role in central

nervous system injury, and Bcl-2 is themost expressed anti-

apoptotic molecule in adult central nervous system.28 The

ratio of proapoptotic to antiapoptotic molecules, such as

Bax/Bcl-2, determines the response to a death signal. In

the present study, we found that pretreatment with

GW0742 reduced Bax expression, whereas increased Bcl-2

expression. Our results also showed that the number of

TUNEL-positive cells was significantly reduced in the

ischemic brain tissue compared with that of the vehicle

group at 24 hours after MCAO by quantitative analysis.

Therefore, the antiapoptotic property of GW0742 might

contribute toabeneficial effect against acute ischemic stroke.

Conclusions

PPAR-b/d agonist GW0742 can protect neuronal dam-

age induced by significant reduction in the infarct

volume, cerebral edema, and the NDSs of rat submitted

to MCAO by reducing inflammatory mediators and

apoptosis. Our results imply that PPAR-b/d agonist

GW0742 may be useful in the therapy of tissue ischemic

injuries.

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Activation of Peroxisome Proliferatoractivated Receptor / Attenuates Acute Ischemic Stroke on Middle Cerebral Ischemia O ...IntroductionMaterials and MethodsAnimalsModel of MCAOExperimental ProtocolEvaluation of Neurologic ImpairmentMeasurement of Infarct VolumeEvaluation of Cerebral EdemaEnzyme-linked Immunosorbent AssayWestern BlotTUNEL AssayStatistical Analysis

ResultsPhysiological ParametersEffects of Pretreatment with GW0742 on Neurologic Deficit and InfarctionEffect of Pretreatment with GW0742 on Brain Edema FormationEffect of Pretreatment with GW0742 on the Expressions of NF-B, TNF-, and IL-1Effect of Pretreatment with GW0742 on Expression of iNOSEffect of Pretreatment with GW0742 on Expressions of Bax and Bcl-2Effect of Pretreatment with GW0742 on in Situ Labeling of DNA Fragmentation

DiscussionConclusionsReferences