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Research Report

Effect of lavender oil (Lavandula angustifolia)on cerebral edema and its possible mechanismsin an experimental model of stroke

Abedin Vakilia,n, Shaghayegh Sharifata, Maziar Mohammad Akhavanb,Ahmad Reza Bandegic

aLaboratory of Cerebrovascular Research, Research Center and Department of Physiology, School of Medicine,Semnan University of Medical Sciences, Semnan, IranbSkin Research Center—Laboratory of Protein and Enzyme, Shahid Beheshti University (M.C.),Shohada-e Tajrish Hospital, Shahrdari St., 1989934148 Tehran, IrancDepartment of Biochemistry, School of Medicine, Semnan University of Medical Sciences, Semnan, Iran

a r t i c l e i n f o

Article history:

Accepted 16 December 2013

Lavender belongs to the family Labiatae and has a variety of cosmetic uses as well as

therapeutic purposes in herbal medicine. The present study was conducted to evaluate the

Available online 30 December 2013

Keywords:

Lavender oil

Brain edema

Focal cerebral ischemia

Oxidative stress

Bcl-2 and Bax

Apoptosis

VEGF

Antioxidant

Rat

nt matter & 2013 Elsevie1016/j.brainres.2013.12.01

hor. Fax: þ98 231 3354186b.vakili@yahoo.com (A. V

a b s t r a c t

protective effect of lavender oil against brain edema and its possible mechanisms in an

experimental model of stroke. Under Laser-Doppler Flowmetry, focal cerebral ischemia was

induced by the transient occlusion of the middle cerebral artery for 1 h in rats. Lavender oil (100,

200, and 400mg/kg ip (and/or vehicle was injected at the onset of ischemia. Infarct size, cerebral

edema, functional outcome, and oxidative stress biomarkers were evaluated using standard

methods. Western blotting was used to determine the protein expression of VEGF, Bax, and

Bcl-2. Treatment with lavender oil at doses of 200 and 400mg/kg significantly diminished

infarct size, brain edema, and improved functional outcome after cerebral ischemia (Po0.001).

Lavender oil (200mg/kg) also reduced the content of malondialdehyde and increased the

activities of superoxide dismutase, glutathione peroxidase, and total antioxidant capacity

(Po0.001). Although lavender oil enhanced VEGF expression (P¼0.026), it could not decrease

the Bax-to-Bcl-2 ratio (pro- to anti-apoptotic proteins) in the rat brain (P40.05). The results

indicated that lavender oil has neuroprotective activity against cerebral ischemia and alleviated

neurological function in rats, and the mechanism may be related to augmentation in

endogenous antioxidant defense, inhibiting oxidative stress, and increasing VEGF expression

in the rat brain. However, lavender oil could not suppress the apoptosis pathway.

& 2013 Elsevier B.V. All rights reserved.

r B.V. All rights reserved.9

.akili).

1. Introduction

Cerebral edema is a life-threatening complication in patientswith stroke, for which no effective treatment has yet been found

(Rosand and Schwamm, 2001). At present, various approachesare used to alleviate brain edema, such as osmosis, diuretics,corticosteroid therapy, and hyperventilation (Rosand andSchwamm, 2001). Despite considerable efforts, a specific therapy

b r a i n r e s e a r c h 1 5 4 8 ( 2 0 1 4 ) 5 6 – 6 2 57

for brain edema has not yet been identified. However, newtherapeutic strategies are needed to more effectively treatbrain edema.

Lavender belongs to the family Labiatae (Lamiacae) and has avariety of therapeutic and cosmetic uses. Traditionally, lavenderwas used as a sedative, carminative, antidepressant, anticonvul-sant, anti-inflammatory, antispasmodic, analgesic, and treatscerebrovascular disease in many nations (Cavanagh andWilkinson 2002; Chu and Kemper 2001; Koulivand et al., 2013).

Lavender is comprised of over 100 constituents, amongwhich the primary components are linalool (51%) and linalylacetate (35%), α-pinene, limonene, 1,8-cineole, cis- and trans-ocimene, 3-octanone, camphor, caryophyllene, terpinen-4-olandlavendulyl acetate, cineole, and flavonoids (Cavanagh andWilkinson, 2002), which are responsible for its pharmacolo-gical activity (Lis-Balchin and Hart, 1999).

Lavender oil is rapidly absorbed by the skin, and theconstituent0s linalool and linalyl acetate are detectable inthe blood 5 min after topical application, peak at 19 min, andlargely disappear from the blood within 90 min (Jager et al.,1992). Very recent evidence suggests that lavender has aprotective effect against ethanol-induced damage in HepG2cells (Farshori et al., in press), scopolamine-induced oxidativestress in the rat brain (Hancianu et al., 2013), and improvedspatial performance in a rat model of Alzheimer disease(Kashani et al., 2011).

There is growing evidence that lavender extract has aprotective effect against cerebral ischemia in vivo (Wang et al.,2012) as well as in vitro (Büyükokuroğlu et al., 2003). However,studies on lavender and cerebral ischemia are limited. Inaddition, the effect of lavender on cerebral edema and itspossible mechanisms have not yet been studied in an experi-mental stroke model. Therefore, the present study aimed toevaluate the effects of lavender oil (Lavandula angustifolia) onischemic lesions, brain edema, neurologic outcome, oxidativestress biomarkers, apoptosis (Bcl-2 and Bax expression), andvascular endothelial growth factor (VEGF) following an ischemicstroke in rats.

Fig. 1 – Regional cerebral blood flow (rCBF; % from baseline)before and during MCAO and after reperfusion in vehicle(MCAOþmaize oil) and lavender oil treated groups at doses of50 (Lav-50), 100 (Lav-100), 200 (Lav-200) and 400 (Lav-400)mg/kg ip.

2. Results

2.1. Local cerebral blood flow

Measurements of local cerebral blood flow (CBF) by Laser-Doppler Flowmetry (LDF) showed that occlusion of the middlecerebral artery caused a reduction in the local CBF to less than20% of baseline during 60min of middle cerebral artery occlusion(MCAO) in all animal groups. Local CBF returned to near baselineafter the suture was removed, but CBF recovered slowly afterreperfusion in 400mg/kg lavender oil treated group. There wasno significant difference in CBF between groups of MCAO (Fig. 1,P40.05). Statistical analysis showed there was a significantdifference in CBF after reperfusion in 400 and 200mg/kg lavenderoil treated groups compared with the vehicle group (Fig.1,P¼0.021).

2.2. Effect of different lavender oil doses on infarct sizeand brain edema

Treatment with lavender oil at doses of 100 and 200 mg/kgsignificantly reduced the infarct volume compared with thevehicle group (Fig. 2A and B; Pr0.001). Lavender oil admin-istration at a dose of 50 mg/kg had no significant effect oninfarct volume (Fig. 2, P40.05).

Induction of focal cerebral ischemia in the vehicle groupsignificantly increased brain water content percent (BWC %)of ischemic hemisphere compared with that in the shamgroup (Po0.001, Fig. 3). Treatment with lavender oil at dosesof 200 and 400 mg/kg significantly reduced the post-ischemicincrease of the BWC% compared with the vehicle group(Po0.001, Fig. 3). No significant difference in the BWC% wasobserved in the non-ischemic hemisphere between thegroups (Fig. 3; P40.05).

2.3. Effect of different doses of lavender oil on neurologicaloutcome

The scores of neurological deficit in lavender oil-treatedgroups at doses of 200 and 400 mg/kg were significantlyimproved compared with the vehicle group (Po0.05, Fig. 4).Lavender oil at a dose of 100 mg/kg did not change theneurological score (P40.05, Fig. 4).

2.4. Effect of lavender oil on activities of SOD, GSH-Pxenzymes, total antioxidant capacity, and MDA content in therat brain

As shown in Table 1, after 1 h MCAO that followed by 23 h ofreperfusion, the activities of superoxide dismutase (SOD) andglutathione peroxidase (GSH-Px) enzymes, and total antiox-idant capacity (TAC) were significantly reduced in the vehiclegroup compared with that in the sham group (Pr0.001).Treatment with lavender oil (200 mg/kg) showed a significantincrease in the activities of SOD, GSH-Px, and total antiox-idant capacity compared with the vehicle group (Pr0.001).

The malondialdehyde (MDA) was used as a biomarker oflipid per-oxidation and oxidative stress. Ischemia caused asignificant increase in the MDA content in the ischemic cortex

Fig. 2 – Infarct volume (A) and TTC staining photograph (B) invehicle (MCAOþmaize oil) and lavender oil treated groups atdoses of 50 (Lav-50), 100 (Lav-100), 200 (Lav-200) mg/kg ip.

Fig. 3 – Brain water content percent (BWC %) in the ischemicand non-ischemic hemispheres in the vehicle (MCAOþmaizeoil), sham-operated and lavender oil treated groups at doses100 (Lav-100), 200 (Lav-200) and 400 (Lav-400) mg/kg ip.

Fig. 4 – Effect of lavender oil at doses of 50 (Lav-50), 100(Lav-100), 200 (Lav-200) and 400 (Lav-400)mg/kg ip on neuro-logical deficits score at 24 h after cerebral ischemia in rats.

b r a i n r e s e a r c h 1 5 4 8 ( 2 0 1 4 ) 5 6 – 6 258

of the vehicle group (Pr0.001).Treatment with lavender oil(200mg/kg) at the onset of ischemia caused a significantreduction in MDA content (Pr0.001; Table 1).

Ischemia causes a significant decrease in Ferric-reducingantioxidant power (FRAP, as a TAC marker) in the brain tissuecompared with the sham group. Treatment with lavender oil(200 mg/kg) at the start of ischemia significantly increasedTAC compared with the vehicle group (Pr0.001; Table 1).

2.5. Effect of lavender oil on the proteins Bcl-2 and Baxand VEGF expression in the rat brain

Induction of cerebral ischemia significantly increases the Bax-to-Bcl-2 ratio (pro- to anti-apoptotic proteins) as a marker ofapoptosis in the brain tissue of vehicle group compared withthe sham group (P¼0.03, Fig. 5). Treatment with lavender oil(200 mg/kg) at the onset of ischemia could not lead to a

significant reduction in the Bax-to-Bcl-2 ratio compared withthe vehicle group (P40.05, Fig. 5).

Western blotting showed that treatment with lavender oil(200 mg/kg) resulted in a significant increase in vascularendothelial growth factor (VEGF) expression (18%) comparedwith the vehicle group (P¼0.026, Fig. 6).

3. Discussion

Infarction and cerebral edema are the two major pathophy-siological changes observed following the acute phase ofstroke. Our study showed that lavender oil, at doses of 100and 200 mg/kg, reduces infarct size by 32% and 47%, respec-tively. Consistent with these results, a recent study reportedthe neuroprotective activity of lavender oil in transient focalcerebral ischemia in mice (Wang et al., 2012). Moreover, for

Table 1 – Effects of lavender oil (200 mg/kg) on the antioxidant enzyme activities (SOD, GSH-Px), FRAP (as a marker of totalantioxidant capacity) and MDA content in the rat brain.

Groups SOD (IU/mg Pr) GSH-Px (U/mg Pr) FRAP (mmol/mg Pr) MDA (nmol/mg Pr)

Sham-operated 9.0870.06 0.2770.02 0.8170.004 4.1370.11Vehicle (MCAOþmaize oil) 7.7870.32# 0.2270.01# 0.7570.01# 5.32727#

Lavender oil (200 mg/kg) 12.3470.22n 0.370.006n 0.8370.003n 3.5711n

Values are mean7SEM.n Po0.05 vs. vehicle.# Po0.05 vs. sham-operated.

Fig. 5 – Bax-to-Bcl-2 ratio in sham-operated, vehicle(MCAOþmaize oil) and lavender oil treated group at dose200 mg/kg (Lav-200) in the rat brain.

Fig. 6 – VEGF expression in sham-operated, vehicle(MCAOþmaize oil) and lavender oil treated group at dose200 mg/kg (Lav-200) in the rat brain.

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the first time, another finding of this research exhibited thatlavender oil attenuated cerebral edema induced by MCAO inrats. It is important to know that cerebral edema is a majorlife-threatening complication in acute phase stroke thatcauses neurological deterioration and or death (Rosand andSchwamm, 2001).These results provided novel evidence ofthe neuroprotective potential of lavender oil in a rat model offocal cerebral ischemia.

Some evidence indicates that lavender oil significantlyattenuated tumor necrosis factor (TNF-α) in isolated rat mastcells (Kim and Cho, 1999). Recent studies from our group andby others have shown that TNF-α, a pro-inflammatory factor,plays an important role in the pathophysiology of cerebraledema and brain injuries after stroke (Vakili et al., 2011; Han,2013). However, based on available evidence (Kim and Cho,1999), a part of the neuroprotective and anti-edematosiseffect observed in this study may be related to the effects oflavender oil in the suppression of TNF-α. Although the TNF-αexpression levels were not measured in this study, further

studies are needed to clarify and confirm this assumption in thefuture. Likewise, it has been reported that the aqueous extractof lavender reduces glutamate-induced neurotoxicity in thecerebellar granular cell culture of rat pups (Büyükokuroğluet al., 2003). We assume that the suppression of glutamatergicneurotoxicity may also be responsible for the neuroprotectiveeffect of lavender oil that was observed in this study.

In most animal studies, the effectiveness of a drug isdetermined by measuring the infarct size (Bae et al., 2013;Schaar et al., 2010). Nevertheless, in clinical trials, neuropro-tective efficacy is measured by neurological function (Schaaret al., 2010), and our present study showed that the neurolo-gical outcome was improved by treatment with lavender oil.Thus, these observations further verify the neuroprotectiveactivity of lavender oil in rats.

Because the brain has high concentrations of peroxidisablelipids, low levels of antioxidative enzymes (SOD, GSH-Px), highoxygen consumption are very susceptible to ROS-induceddamage following ischemia–reperfusion, which causes oxidativedamage to brain lipids, proteins, and DNA, finally leading to

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brain dysfunction and cell death (Murakami et al., 1997; Allenand Bayraktutan, 2009). Since reactive oxygen species (ROS) has ashort half-life, its direct measurement in the brain is fairlydifficult (Allen and Bayraktutan, 2009). MDA level is commonlyused as a biomarker of oxidative stress in in vivo studies (Allenand Bayraktutan, 2009). MDA is a peroxidation product ofpolyunsaturated fatty acids in cells that are primarily inducedby ROS (Allen and Bayraktutan, 2009). Our study showed thatlavender oil reduced the post-ischemic-enhanced MDA contentas a biomarker of oxidative stress, and increased the activities ofSOD, GSH-Px as major antioxidant enzymes. This result corro-borates the result of another study (Wang et al., 2012; Hancianuet al., 2013). Our findings suggested that lavender oil attenuatedthe cerebral ischemia–reperfusion injury in rats by over expres-sion and augmentation of antioxidant enzymes and by suppres-sing the oxidative stress pathway.

Apoptosis is one of the most important mechanisms thatlead to neuronal death after cerebral ischemia (Chelluboinaet al., in press; Ferrer, 2006).The Bcl-2-associated X protein (BAX)a protein of the Bcl-2 gene family that promotes apoptosis bycompeting with Bcl-2 as an anti-apoptotic protein (Raghupathiet al., 2003; Chan, 2004). Our results showed occlusion of MCAOsignificantly increased apoptosis (Bax-to-Bcl-2 ratio) in theischemic brain tissue compared with the sham-operated group,but treatment with lavender oil could not reduce the apoptosisafter cerebral ischemia in the rat brain. However, our results arein contrast with a recent study indicating that inhaled lavenderoil promotes anti-apoptotic activity in a scopolamine-induceddementia rat model (Hancianu et al., 2013). In this study(Hancianu et al., 2013), lavender oil inhaled for 7 days andapoptosis was measured with DNA fragmentation assays in ascopolamine-induced dementia rat model, while in our presentstudy, effects of lavender oil (200mg/kg ip) were assessed in atransient model of focal cerebral ischemia and apoptosis,evaluated using western blotting. These results indicated thatthe apoptosis pathwaysmay not be involved in neuroprotectionby lavender oil that was observed in the present study. Thisfinding suggests that other mechanisms may also contribute.

Another finding of our study indicated that lavender oilinduced VEGF expression in the rat brain during the acute phaseof stroke. It appears that at least a part of the neuroprotectiveeffect of lavender oil that was observed in this study mayberelated to increased VEFG expression in the ischemic area. Themechanisms underlying VEGF-induced reduction of ischemicinjury are not clear in the present study. VEGF is inducedfollowing focal cerebral ischemia and promotes the formationof new cerebral blood vessels in the ischemic area (Rosensteinet al., 1998; Sun et al., 2003). Revascularization in the brain infarctusually occurred at 2–5 days after stroke and continued forseveral months (Hayashi et al., 1997; Sun et al., 2003). Since thecurrent study ischemic injury was determined 24 h after stroke,the neuroprotective effect of VEGF may be independent of theangiogenesis function and may be related to other mechanisms.VEGF protects capillary endothelial cells from apoptotic celldeath (Alon et al., 1995) and also relaxes vascular smoothmusclecells (Ku et al., 1993). Thus, part of the direct neuroprotectiveactivity of VEGF may be associated with these effects.

In conclusion, the present study showed that lavender oilreduced the brain infarct size, edema, and improved theneurological function in a rat model of focal ischemic stroke.

The protective effects of lavender oil were mediated throughthe augmentation of endogenous antioxidant defenses, inhi-bition of the oxidative stress pathway, and increased VEGFexpression in the rat brain. Moreover, lavender oil did notexhibit anti-apoptotic effects in this study. These findingssuggest that lavender oil may have immense potential andprove to be a beneficial agent for the treatment of strokepatients.

4. Experimental procedures

4.1. Animals

Adult male Wistar rats (300720 g) were obtained from thebreeding colony of Semnan University of Medical Sciences(SUMS), Semnan, Iran. Animals were housed in polyacryliccages, with not more than four rats per cage under standardlaboratory conditions, under natural light and dark cycles(approximately 12 h light/12 h dark), at a temperature of2472 1C. They were fed a pellet diet and tap water, ad libitum.All procedures were performed in accordance with theNational Institutes of Health Guide for Care and Use ofLaboratory Animals.

4.2. Surgical procedure and measurement of CBF

Transient focal cerebral ischemia was induced under chloralhydrate (400 mg/kg, Merck, Germany) anesthesia in rats(Vakili et al., 2011; Longa et al., 1989). A 3-0 nylon suturewas introduced into the internal carotid artery and gentlyadvanced until LDF (Moor Instruments DRT4, UK) showed asharp decrease in the ipsilateral CBF to less than 20% ofbaseline, which confirmed an occlusion of the middle cere-bral artery. After 60 min of middle cerebral artery occlusion(MCAO), reperfusion was started by withdrawing the nylonthread for 23 h. To measure CBF, an LDF probe was positionedin direct contact with the right temporal bone after a limiteddissection of the temporalis muscle at an equal distancebetween the eye and ear (Lecrux et al., 2007). To fix andprevent the displacement of the LDF probe, a burr hole (2-mmdiameter) was drilled 5 mm lateral and 1 mm posterior to thebregma without injury to the dura mater. CBF was continu-ously monitored 15 min before, during MCAO, and up to15 min after the reperfusion.

4.3. Neurobehavioral test

The neurobehavioral (sensory and motor) test was performed24 h after MCAO as described previously (Reglodi et al., 2003).The sum of partial scores yielded the total neurological score,with a maximum of 42 points and a minimum of 0 in normalrats (Reglodi et al., 2003). The neurological score of rats wasevaluated blindly by an investigator who was unaware of theanimal groups.

4.4. Measurement of infarct size

After neurological evaluation, rats were sacrificed with ananesthesia overdose and the brains were rapidly excised. The

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brain was coronally sectioned into seven 2-mm thick slicesusing a brain matrix. Coronal sections were stained in 2%2,3,5-triphenyltetrazolium chloride (TTC; Sigma, Germany) at37 1C in a water bath for 15 min, and then fixed in 4%paraformaldehyde for 24 h. The brain slices were then photo-graphed separately using a digital camera (Cannon-Japan).Infarct areas of all sections were measured using an imageanalysis software (NIH Image Analyzer). The infarct volumeof each slice was calculated by multiplying the infarct area ofthe slice by its thickness. The injury volume was calculatedby multiplying the infarct area by the thickness of the brainsections. The infarct volume was adjusted for edema.

4.5. Measurement of cerebral edema

Cerebral edema was evaluated by determining the BWC (Vakiliet al., 2011). Twenty-four hours after MCAO, the rats weresacrificed and their brains were excised. Subsequently, the Ponsand olfactory bulb were removed; the brains were weighed toobtain their wet weight (WW), and dried at 110 1C for 24 h todetermine their dry weight (DW). BWC was calculated using thefollowing formula: (WW�DW)/WW�100.

4.6. Oxidative stress marker assay

Following cerebral ischemia, the whole ischemic hemispherewas homogenized (1:10 w/v) in cold 1.15% KCl and centrifuga-tion at 20,000g at 4 1C for 10 min. The supernatants were usedfor biochemical analyses. The total protein level in thesupernatants was determined by the Bradford method usingbovine serum albumin as standard (Bradford, 1976). The MDAcontent or thiobarbituric acid reactive substances (TBARS)were measured using the thiobarbituric acid method asdescribed previously by Mihara and Uchiyama (1978). SODand GSH-Px activities were measured using a commercial kit(Randox Laboratories Ltd., UK) and according to man-ufacturer0s protocol. FRAP assay is a colorimetric methodfor measuring the TAC (Benzie and Strain, 1999).

4.7. Protein measurement and western blotting

Brain samples were homogenized and prepared in lysis buffer(137 mM NaCl, 20 mM Tris–HCl pH 8.0, 1% NP-40, 10% glycerol,1 mM phenylmethyl sulfonyl fluoride, 10 μg/ml aprotinin,1 μg/ml leupeptin, and 0.5 mM sodium vanadate). Tissueextracts were centrifuged to remove insoluble material(12500g for 20 min at 4 1C) and total protein concentrationwas determined according to the Micro BCA procedure(Pierce, Rockford, IL, USA). Equal amounts (25 μg) of proteinfrom each sample were loaded on 15% polyacrylamide gelsand separated by a standard SDS-PAGE. Protein bands weretransferred to PVDF membranes and then blocked using 5%skim milk and 0.1% Tween-20 in Tris-buffered saline. Mem-branes were incubated with primary antibodies overnight at4 1C [anti-Bax, 1:500 (sc-493); anti Bcl-2, 1:200 (sc-492) anti-actin, 1:300 (sc-130065); anti VEGF, 1:1000 (sc-152)] and thenwith a secondary antibody [(goat anti-rabbit IgG horseradishperoxidase conjugated antibody, 1:2000 (sc-2004)] for 1 h atroom temperature. Immunocomplexes were visualized bychemiluminescence using the ECL kit (Pierce, Rockford, IL,

USA) according to the manufacturer0s instructions. Theresults for target proteins, Bax, Bcl-2, VEGF, and actin werequantified by densitometric analysis (using Gel-Pro analyzerimaging software). The results for the Bax-to-Bcl-2 ratio werecalculated and the results for VEGF were normalized for beta-actin levels.

4.8. Experimental design and treatment plan

To determine the effect of lavender oil (Sigma-Aldrich,Germany) on infarct size and neurological outcome, 28animals were randomly divided into four experimentalgroups, including vehicle (MCAOþmaize oil, 1 ml/kg, n¼7)and treatment groups in which lavender oil was administeredintraperitoneally at doses of 50 mg/kg (n¼7), 100 mg/kg (n¼7),and 200 mg/kg (n¼7), at the onset of MCAO.

To evaluate the therapeutic effect of lavender oil on brainedema, we used five different groups: sham operated (n¼7),vehicle (MCAOþmaize oil, 1 ml/kg, n¼7), and lavender oil atdoses 100 mg/kg (n¼7), 200 mg/kg (n¼7), 400 mg/kg (n¼7),administered at onset of MCAO.

To determine the effect of lavender oil on MDA content,SOD and GSH-Px activities, and the total antioxidant capacityin the ischemic hemisphere, 18 animals were randomlydivided into three experimental groups, including shamoperated (n¼6), vehicle (MCAOþmaize oil, 1 ml/kg, n¼6),and lavender oil (200 mg/kg, n¼6)-treated, administered atthe onset of MCAO.

To determine the effect of lavender oil on the expressionof Bcl-2 (anti-apoptotic protein) Bax (pro-apoptotic protein),and VEGF protein in the rat brain, 18 animals were randomlydivided into three experimental groups, including shamoperated (n¼6), vehicle (MCAOþmaize oil, 1 ml/kg, n¼6),and lavender oil (200 mg/kg, n¼6)-treated, administered atthe onset of MCAO.

4.9. Statistical analysis

All results were presented as mean7SEM. Statistical analysiswas conducted using Kruskal–Wallis one-way analysis ofvariance (ANOVA) followed by the Dunn0s test and ANOVAfollowed by the Holm–Sidak method as post hoc analysis.Differences were considered significant at Po0.05 (SigmaStat2.0; Jandel Scientific, Erkrath, Germany).

Acknowledgments

This work was financially supported (Grant no. 434) by theVice Chancellor for Research Centers of Semnan University ofMedical Sciences, Semnan, Iran. This article is extracted fromthe MS.C. student thesis of Shaghayegh Sharifat.

r e f e r e n c e s

Allen, C.L., Bayraktutan, U., 2009. Oxidative stress and its role inthe pathogenesis of ischaemic stroke. Int. J. Stroke 4, 461–470.

Alon, T., Hemo, I., Itin, A., Pe0er, J., Stone, J., Keshet, E., 1995.Vascular endothelial growth factor acts as a survival factor for

b r a i n r e s e a r c h 1 5 4 8 ( 2 0 1 4 ) 5 6 – 6 262

newly formed retinal vessels and has implications forretinopathy of prematurity. Nat. Med. 1, 1024–1028.

Bae, O.N., Serfozo, K., Baek, S.H., Lee, K.Y., Dorrance, A.,Rumbeiha, W., Fitzgerald, S.D., Farooq, M.U., Naravelta, B.,Bhatt, A., Majid, A., 2013. Safety and efficacy evaluation ofcarnosine, an endogenous neuroprotective agent for ischemicstroke. Stroke 44, 205–212.

Benzie, I.F., Strain, J.J., 1999. Ferric reducing/antioxidant powerassay: direct measure of total antioxidant activity of biologicalfluids and modified version for simultaneous measurement oftotal antioxidant power and ascorbic acid concentration.Methods Enzymol. 299, 15–27.

Bradford, M.M., 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal. Biochem. 72, 248–254.

Buyukokuroglu, M.E., Gepdiremen, A., Hacimuftuoglu, A., Oktay,M., 2003. The effects of aqueous extract of Lavandulaangustifolia flowers in glutamate-induced neurotoxicity ofcerebellar granular cell culture of rat pups. J. Ethnopharmacol.84, 91–94.

Cavanagh, H.M., Wilkinson, J.M., 2002. Biological activities oflavender essential oil. Phytother. Res. 16, 301–308.

Chan, P.H., 2004. Mitochondria and neuronal death/survivalsignaling pathways in cerebral ischemia. Neurochem. Res. 29,1943–1949.

Chelluboina B., Klopfenstein J.D., Gujrati M., Rao J.S., Veeravalli K.K.Temporal regulation of apoptotic and anti-apoptoticmolecules after middle cerebral artery occlusion followed byreperfusion. Mol. Neurobiol. http://dx.doi.org/10.1007/s12035-013-8486-7, in press.

Chu, C.J., Kemper, K.J., 2001. Lavender ( Lavandula spp.), TheLongwood Herbal Task Force (http://www.mcp.edu/herbal/)and The Center for Holistic Pediatric Education and Research(http://www.childrenshospital.org/holistic/). Available at:http:// www.longwoodherbal.org/ lavender/lavender.pdf.

Farshori N.N., Al-Sheddi E.S., Al-Oqail M.M., Hassan W.H., Al-Khedhairy A.A., Musarrat J. and Siddiqui M.A.,Hepatoprotective potential of Lavandula coronopifoliaextracts against ethanol induced oxidative stress-mediatedcytotoxicity in HepG2 cells, Toxicol. Ind. Health. http://dx.doi.org/10.1177/0748233713483188, in press.

Ferrer, I., 2006. Apoptosis: future targets for neuroprotectivestrategies. Cerebrovasc. Dis. 21 (suppl. 2), 9–20.

Han, T., 2013. Effects of salidroside pretreatment on expression oftumor necrosis factor-alpha and permeability of blood brainbarrier in rat model of focal cerebral ischemia-reperfusioninjury. Asian Pac. J. Trop. Med. 6, 156–158.

Hancianu, M., Cioanca, O., Mihasan, M., Hritcu, L., 2013.Neuroprotective effects of inhaled lavender oil onscopolamine-induced dementia via anti-oxidative activities inrats. Phytomedicine 20, 446–452.

Hayashi, T., Abe, K., Suzuki, H., Itomaya, Y., 1997. Rapid inductionof vascular endothelial growth factor gene expression aftertransient middle cerebral artery occlusion in rats. Stroke 28,2039–2044.

Jager, W., Buchbauer, G., Jirovetz, L., Fritzer, M., 1992.Percutaneous absorption of lavender oil from massage oil. J.Soc. Cosmet. Chem. 43, 49–54.

Kashani, M.S., Tavirani, M.R., Talaei, S.A., Salami, M., 2011.Aqueous extract of lavender (Lavandula angustifolia)

improves the spatial performance of a rat model ofAlzheimer0s disease. Neurosci. Bull. 27, 99–106.

Kim, H.M., Cho, S.H., 1999. Lavender oil inhibits immediate-typeallergic reaction in mice and rats. J. Pharm. Pharmacol. 51,221–226.

Koulivand P.H., Khaleghi Ghadiri M. and Gorji A., Lavender andthe nervous system, EvidEvid. Based Complement AlternatMed. Alternat. Med. 2013 (2013), Article ID 681304, 10 pp,http://dx.doi.org/10.1155/2013/681304.

Ku, D.D., Zaleski, J.K., Liu, S., Brock, T.A., 1993. Vascularendothelial growth factor induces EDRF-dependent relaxationin coronary arteries. Am. J. Physiol. 265, H586–H592.

Longa, E.Z., Weinstein, P.R., Carlson, S., Cummins, R., 1989.Reversible middle cerebral artery occlusion withoutcraniectomy in rats. Stroke 20, 84–91.

Lecrux, C., Nicole, O., Chazalviel, L., Catone, C., Chuquet, J.,MacKenzie, E.T., Touzani, O., 2007. Spontaneouslyhypertensive rats are highly vulnerable to AMPA-inducedbrain lesions. Stroke 38, 3007–3015.

Lis-Balchin, M., Hart, S., 1999. Studies on the mode of action ofthe essential oil of lavender (Lavandula angustifolia P. Miller).Phytother. Res. 13, 540–542.

Mihara, M., Uchiyama, M., 1978. Determination of malonaldehydeprecursor in tissues by thiobarbituric acid test. Anal. Biochem.86, 271–278.

Murakami, K., Kondo, T., Chan, P.H., 1997. Reperfusion followingfocal cerebral ischemia alters distribution of neuronal cellswith DNA fragmentation in mice. Brain Res. 751, 160–164.

Raghupathi, R., Strauss, K.I., Zhang, C., Krajewski, S., Reed, J.C.,McIntosh, T.K., 2003. Temporal alterations in cellular Bax:Bcl-2ratio following traumatic brain injury in the rat. J.Neurotrauma 20, 421–435.

Reglodi, D., Tamas, A., Lengvari, I., 2003. Examination ofsensorimotor performance following middle cerebral arteryocclusion in rats. Brain Res. Bull. 59, 459–466.

Rosand, J., Schwamm, L.H., 2001. Management of brain edemacomplicating stroke. J. Intensive Care Med. 16, 128–141.

Rosenstein, J.M., Mani, N., Silverman, W.F., Krum, J.M., 1998.Patterns of brain angiogenesis after vascular endothelialgrowth factor administration in vitro and in vivo. Proc. Natl.Acad. Sci. U. S. A 95, 7086–7091.

Schaar, K.L., Brenneman, M.M., Savitz, S.I., 2010. Functionalassessments in the rodent stroke model. Exp. Transl. StrokeMed. 19 (2(1)), 13, http://dx.doi.org/10.1186/2040-7378-2-13.

Sun, Y., Jin, K., Xie, L., Childs, J., Mao, X.O., Logvinova, A.,Greenberg, D.A., 2003. VEGF-induced neuroprotection,neurogenesis, and angiogenesis after focal cerebral ischemia.J. Clin. Invest. 111, 1843–1851.

Vakili, A., Mojarad, S., Akhavan, M.M., Rashidy- pour, A., 2011.Pentoxifylline attenuates TNF-α protein levels and brainedema following temporary focal cerebral ischemia in rats.Brain Res. 1377, 119–125.

Wang, D., Yuan, X., Liu, T., Liu, L., Hu, Y., Wang, Z., Zheng, Q.,2012. Neuroprotective activity of lavender oil on transientfocal cerebral ischemia in mice. Molecules 17, 9803–9817.