Table Of ContentQuartuetal.LipidsinHealthandDisease2012,11:8
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RESEARCH Open Access
Pistacia lentiscus
Effect of acute administration of
L. essential oil on rat cerebral cortex following
transient bilateral common carotid artery
occlusion
Marina Quartu1*†, Maria P Serra1†, Marianna Boi1, Giuliano Pillolla1, Tiziana Melis1, Laura Poddighe1,
Marina Del Fiacco1, Danilo Falconieri2, Gianfranca Carta1,3, Elisabetta Murru1,3, Lina Cordeddu1,3, Antonio Piras1,3,
Maria Collu1 and Sebastiano Banni1,3
Abstract
Background: Ischemia/reperfusion leads to inflammation and oxidative stress which damages membrane highly
polyunsaturated fatty acids (HPUFAs) and eventually induces neuronal death. This study evaluates the effect of the
administration of Pistacia lentiscus L. essential oil (E.O.), a mixture of terpenes and sesquiterpenes, on modifications
of fatty acid profile and endocannabinoid (eCB) congener concentrations induced by transient bilateral common
carotid artery occlusion (BCCAO) in the rat frontal cortex and plasma.
Methods: Adult Wistar rats underwent BCCAO for 20 min followed by 30 min reperfusion (BCCAO/R). 6 hours
before surgery, rats, randomly assigned to four groups, were gavaged either with E.O. (200 mg/0.45 ml of
sunflower oil as vehicle) or with the vehicle alone.
Results: BCCAO/R triggered in frontal cortex a decrease of docosahexaenoic acid (DHA), the membrane highly
polyunsaturated fatty acid most susceptible to oxidation. Pre-treatment with E.O. prevented this change and led
further to decreased levels of the enzyme cyclooxygenase-2 (COX-2), as assessed by Western Blot. In plasma, only
after BCCAO/R, E.O. administration increased both the ratio of DHA-to-its precursor, eicosapentaenoic acid (EPA),
and levels of palmytoylethanolamide (PEA) and oleoylethanolamide (OEA).
Conclusions: Acute treatment with E.O. before BCCAO/R elicits changes both in the frontal cortex, where the
BCCAO/R-induced decrease of DHA is apparently prevented and COX-2 expression decreases, and in plasma, where
PEA and OEA levels and DHA biosynthesis increase. It is suggested that the increase of PEA and OEA plasma levels
may induce DHA biosynthesis via peroxisome proliferator-activated receptor (PPAR) alpha activation, protecting
brain tissue from ischemia/reperfusion injury.
Keywords: Bilateral common carotid artery occlusion, reperfusion, DHA; COX-2, PEA, OEA, Pistacia lentiscus L., cere-
bral cortex, Wistar rat
*Correspondence:[email protected]
†Contributedequally
1DepartmentofBiomedicalSciences,UniversityofCagliari,Cittadella
Universitaria,Monserrato,Italy
Fulllistofauthorinformationisavailableattheendofthearticle
©2012Quartuetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
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Background supported by different experimental data [19,21-25]. The
Cerebral ischemia or stroke is often associated to unilat- Pistacia lentiscus L. plant is an aromatic bush indigenous
eral or bilateral occlusion of internal carotid artery or to Italy and other Mediterranean and Middle East coun-
common carotid artery [1-4]. Interruption of brain cir- tries. Its extracts have found extensive use in folk medi-
culation makes the cerebral tissue unable to sustain cine for their anti-hypertensive, anti-inflammatory and
basal metabolism and within a few seconds leads to antiseptic properties and for the treatment of gastric dis-
multiple interconnected pathophysiological events affect- orders [26]. To our knowledge, the bioactive properties
ing the structural and functional organization of brain of P. lentiscus L. extracts and/or individual components
tissue [5,6]. have been reported only by in vitro studies where, with
Although reperfusion of neural ischemic tissue is different analytical methods, it has been shown that they
desirable, the post-ischemic reestablishment of blood have a strong antioxidant activity [25,27-33]. Previous
supply leads to a general impairment of translation cap- analyses of P. lentiscus L. essential oil (E.O.) showed a
ability, oxidative stress and free radical formation which characteristic composition with relatively high concentra-
can eventually cause neuronal death [6-8]. Neuronal tion of terpenes and sesquiterpenes [27-33], for some of
membranes are particularly rich in highly polyunsatu- which a potent anti-inflammatory activity has been
rated fatty acids (HPUFAs) which serve as reservoirs of shown in different experimental rodent models
biologically active lipids in physiological conditions, [21,22,34-36].
whereas, in stressful circumstances, are target of free Because of the low extraction yield from P. lentiscus L.
radical-mediated lipid peroxidation whose products, in plant, the E.O. under study has not generally received
turn, can injure the brain [9-11]. The brain has a unu- much attention for its application in pharmaceutics and
sual ability to preserve the neuronal membrane concen- has little commercial use. We sought to investigate the
tration of HPUFAs within a physiological range [12]; potentially neuroprotective in vivo effects of dietary E.
however, it is also particularly vulnerable to oxidative O., obtained by Supercritical Fluid Extraction (SFE), in a
stress due to the presence of uniquely high content of rat model of hypoperfusion achieved by transient bilat-
HPUFAs, of an inadequate antioxidant defense system eral common carotid artery occlusion (BCCAO) fol-
and of high mitochondrial density [10,11]. Ischemia/ lowed by reperfusion (BCCAO/R).
reperfusion may affect lipid turnover and cause the This study reports on modifications of fatty acid pro-
release of free (unesterified) PUFAs from membrane file and eCB congener concentrations induced by
phospholipids [9]. The main products of lipid peroxida- BCCAO/R in the rat frontal cortex and plasma, and
tion are fatty acid hydroperoxides which are quite shows that lipid metabolic changes are apparently pre-
unstable and capable to propagate free radical reactions, vented by the administration of E.O.. Expression of the
thus extending the damage [13]. enzyme cyclooxygenase-2 (COX-2) is also examined by
Interestingly, recent studies point up the relationship Western Blot on the same brains.
between brain metabolic stress and lipid peripheral dys-
regulation, showing that cleavage and immediate release Methods
of membrane lipid precursors, such as eicosanoids, Extraction of P. lentiscus L. essential oil
could be useful indicators of central nervous system Plant material
(CNS) pathologies [14]. In addition, plasma lipid meta- Leaves of P. lentiscus L. were collected during full blos-
bolic alterations as a physiological response to brain som in northern Sardinia (Italy).
ischemic stimulus are triggered by ischemia/reperfusion Reagents and standards
during carotid endoarterectomy in patients with carotid Highest purity solvents and reagents included Folin-
stenosis [15] and endocannabinoid (eCB) plasma levels Ciocalteu reagent, methanol, hydrochloric acid (37%),
have been shown to increase in patients with acute ferric chloride, sodium acetate trihydrate and sodium
ischemic stroke [16]. acetate 6-hydrate (Merck, Darmstadt, Germany);
Epidemiological studies indicate that oxidative stress 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH, 98%) and
and neuroinflammation are also factors associated with gallic acid (99%) (Sigma-Aldrich, Steinheim, Germany);
the decline of function in the aging brain [17] and that sodium carbonate anhydrous and 2,4,6-tris(2-pyridyl)-
the regular consumption of natural products rich in S-triazine (TPTZ, P99%) (Fluka, Buchs, Germany); gla-
bioactive compounds is associated with vascular diseases, cial acetic acid (SDS, Penien, France); dichloromethane
stroke and dementia risk reduction [18-22]. Although the (Lab-Scan, Dublin, Ireland). Chemical constituents of
mechanisms through which these compounds exert bene- the oil were identified by comparison with reference
ficial effects wait to be clarified, their antioxidant, free compounds [Fluka, Acros Organics (Geel, Belgium);
radical scavenging and anti-inflammatory properties are Sigma-Aldrich].
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Preparation of the extracts Table 1Retention indices,I andchromatographic area
R
Supercritical CO (SFE) extractions were performed in a percentages (%) ofcompounds foundin E.O. extracted
2
laboratory apparatus, equipped with a 320 cm3 extrac- by SFEat 90bar, 50°CfromPistacialentiscus L.leaves
tion vessel and two separator vessels of 300 and 200 n. I Compound %
k
cm3, respectively, connected in series [37]. Experiments 1 922 tricyclene -
were carried out at P = 90 bars, T = 50°C and j =
CO2 2 926 alpha-thujene 0.2
0.6 kg/h. In the first separator the temperature was set
3 933 alpha-pinene 6.3
at -10°C and the pressure at the same value as the
4 948 camphene 0.3
extraction section. The second separator was set at 10°C
5 973 sabinene 5.8
and 15 bars. Extraction was carried out in a semi batch
6 977 beta-pinene 2.4
mode: batch charging of vegetable matter and continu-
ous flow solvent. About 180 g of material were charged 7 991 myrcene 3.9
in each run. 8 1006 alpha-phellandrene 1.5
Analysis 9 1016 alpha-terpinene 0.4
Analysis of essential oil was carried out by gas chroma- 10 1024 para-cymene 0.8
tography (GC) and by gas chromatography-mass spec- 11 1028 beta-phellandrene 3.7
trometry (GC-MS). Analytical GC was carried out in an
12 1046 (E)-beta-ocimene -
Agilent 6890 gas chromatograph (Agilent Technologies,
13 1054 isopentiln-butanoate 0.2
Palo Alto, CA, USA) with HP GC ChemStation data
14 1057 gamma-terpinene 0.7
handling system, equipped with a single injector and
15 n.i. 0.2
two flame ionization detectors (FID). A Graphpak divi-
16 1088 terpinolene 0.2
der (Agilent Technologies) was used for simultaneous
sampling to two Supelco fused silica capillary columns 17 1091 2-nonanone 0.2
(Supelco Inc., Bellefonte, PA, USA) with different sta- 18 n.i. 0.1
tionary phases: SPB-1 (polydimethylsiloxane 30 m × 19 1101 linalool 0.2
0.20 mm I.D., film thickness 0.20 μm) and SUPELCO- 20 1106 isopentylisovalerate -
WAX 10 (polyethylene glycol 30 m × 0.20 mm I.D., film 21 1177 terpin-4-ol 1.0
thickness 0.20 μm). Oven temperature was settled at 70°
22 1190 alpha-terpineol 0.2
C, raising at 3°C min-1 to 220°C and then held 15 min
23 1250 n.i. 0.4
at 220°C; injector temperature: 250°C; carrier gas:
24 1253 n.i. 0.2
helium, adjusted to a linear velocity of 30 cm/s; splitting
25 1285 bornylacetate -
ratio 1:40; detector temperature: 250°C.
GC-MS analyses were carried out in an Agilent 6890 26 1294 2-undecanone 1.0
gas chromatograph fitted with a HP1 fused silica col- 27 1302 n.i. -
umn (polydimethylsiloxane 30 m × 0.25 mm I.D., film 28 1349 alpha-cubebene 0.2
thickness 0.25 μm), interfaced with an Hewlett Packard 29 1375 alpha-copaene 1.7
mass selective detector 5973 (Agilent Technologies) 30 1389 beta-cubebene 1.1
operated by Agilent Enhanced ChemStation software.
31 1391 beta-elemene 1.3
GC parameters as above; interface temperature: 250°C;
32 1408 n.i. -
MS source temperature: 230°C; MS quadrupole tem-
33 1418 beta-caryophyllene 6.6
perature: 150°C; ionization energy: 70 eV; ionization
current: 60 μA; scan range: 35-350 u; scans/sec: 4.51. 34 1428 beta-gurjunene 0.2
35 1436 n.i. 0.7
The identity of the components was assigned by com-
parison of mass spectra and retention indices for two 36 1449 n.i. 0.3
different chromatographic stationary phases calculated 37 1452 alpha-humulene 2.4
by linear interpolation to the retention of a series of n- 38 1459 allo-aromadendrene 0.9
alkanes. Experimental data were compared with corre- 39 1461 cis-muurola-4(14),5-diene -
sponding data of reference oils and commercial available 40 1473 trans-cadina-1(6),4-diene -
standards banked at a home-made library or from litera-
41 1477 gamma-muurolene 2.9
ture data [38,39]. Percentages of individual components
42 1481 germacreneD 19.9
were calculated based on GC peak areas without FID
43 1485 beta-selinene -
response factor correction.
44 1490 trans-muurola-4(15),5-diene 0.3
Composition of the E.O. is reported in Table 1. The
45 n.i. 1.9
total essential oil yield, after an extraction lasting 4
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Table 1 Retention indices, I and chromatographic area handling throughout the experimental procedures were
R
percentages (%) of compounds found in E.O. extracted in accordance with the European Communities Council
by SFE at 90 bar, 50?°?C from Pistacia lentiscus L. leaves Directive of 24 November 1986 (86/609/EEC). The
(Continued)
experimental protocols were also approved by the Ani-
46 1495 alpha-selinene 1.7 mal Ethics Committee of the University of Cagliari.
Standard laboratory food (Safe A04, France) and water
47 1499 alpha-muurolene 1.6
were freely available ad libitum.
48 1503 n.i. 0.9
Animals were starved for 12 hours before surgery and
49 1506 n.i. -
6 hours prior to ischemia the E.O. was administered by
50 1509 beta-bisabolene 1.1
gavage. Due to the lack of data in the literature on the
51 1513 gamma-cadinene 8.7 use of E.O. in vivo, choice of the dose was based on the
52 n.i. 0.2 concentration of some of the E.O. components, such as
53 1524 delta-cadinene 4.2 BCP and alpha-humulene (Table 1), whose effectiveness
54 1531 trans-cadina-1(2)-4-diene 0.5 as single agents has been reported [34-36]. Since these
55 1536 alpha-cadinene - molecules have been shown to be active in contrasting
56 1543 n.i. 0.4 the symptoms of different models of inflammation in
the range of 5 to 100 mg/kg bodyweight [34-36], in the
57 1548 elemol 0.4
present study each rat was administered 200 mg of E.O.
58 1555 n.i. 0.3
(containing about 60 mg/kg bodyweight of BCP and 22
59 1557 elemicin 0.4
mg/kg bodyweight of alpha-humulene) in 0.45 ml of
60 1564 (E)-nerolidol -
sunflower oil as vehicle. Rats were randomly assigned to
61 n.i. 0.7
four groups, those submitted to BCCAO/R and the
62 1575 spatutenol 1.8 sham-operated ones, treated with the vehicle alone or
63 1581 caryophylleneoxide 0.2 with the vehicle containing the E.O..
64 1583 n.i. 0.2 Surgery
65 1589 n.i. 0.2 Surgery was performed in all cases between 15:00 and
17:30 p.m.. Rats were anesthetized with intraperitoneal
66 1613 n.i. -
administration of chloral hydrate (80 mg/ml; 0.5 ml/
67 1615 n.i. -
100 g bodyweight). After a midline cervical incision
68 1626 n.i. -
and blunt dissection of muscles, the right and left com-
69 1630 gamma-eudesmol -
mon carotid arteries (CCA) were exposed while leaving
70 1641 epi-alpha-muurolol 1.0
the vagus nerve intact. Transient bilateral ligation of
71 1645 alpha-muurolol 0.4 CCA, performed with a 0.28 mm nylon fishing line,
72 1648 beta-eudesmol - lasted 20 min. Blood flow was then restored through
73 1653 alpha-cadinol 0.9 the stenosed vessels for 30 min. Sham operated rats
74 1682 epi-alpha-bisabolol 0.7 underwent a surgical procedure similar to the other
75 1684 alpha-bisabolol 0.3 groups but without the CCA occlusion. These animals
made up the control group used to determine the
76 1694 n.i. 3.1
effects of anaesthesia and surgical manipulation on the
results.
hours, was 0.14% and the main constituents included Sampling
germacrene D (19.9%), beta-caryophyllene (BCP) (6.6%), At the end of the procedure, the animals, still anesthe-
alpha-pinene (6.3%), myrcene (3.9%), beta-phellandrene tized, were sacrificed and different brain areas, i.e. olfac-
(3.7%) and alpha-humulene (2.4%). tory bulb, telencephalic cortex (subdivided in an
anterior third, including the frontal cortex, and the
Experimental procedure remaining posterior two thirds, including parietal, tem-
Animals and keeping poral, and occipital cortex), hippocampus, hypothala-
Sixteen male Wistar rats (Harlan-Italy, Udine, Italy ), mus, striatum, brainstem and cerebellum were rapidly
weighing 210 ± 20 g (mean ± SD) were housed in an dissected out and frozen at - 80°C until fatty acid or
environment of controlled temperature (21 ± 2°C), rela- western blot analysis. Blood was collected from the
tive humidity (60 ± 5%) and under an artificial 12 h trunk of killed rats into heparinised tubes and centri-
light/dark cycle for 1 week before the experiment set fuged at 1500 g for 10 min at - 8°C. The resulting
off. All stressful stimuli were avoided. Animal care and plasma was frozen at - 20°C until assayed for lipids.
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Lipid extraction 1:5,000, as secondary antiserum. In order to control for
Measurement of fatty acid composition non specific staining, blots were stripped and incubated
Total lipids were extracted from different brain areas with the relevant secondary antiserum. After TBS-T
and plasma using chloroform/methanol 2:1 (v/v) [40]. rinse, protein bands were visualized on a film (Kodak X-
Aliquots were mildly saponified as previously described Omat LS, Kodak, Rochester, NY) using the ECL method
[41] in order to obtain free fatty acids for high-perfor- (Amersham Corp.). Approximate molecular weight
mance liquid chromatography (HPLC) analysis. Separa- (mw) of immunolabelled protein bands and relative
tion of fatty acids was carried out with an Agilent 1100 optical densities (ODs) were quantified using a GS-
HPLC system (Agilent, Palo Alto, Calif., USA) equipped 800™ Calibrated Densitometer with Quantity One 1
with a diode array detector as previously reported [42]. analysis software (BIO-RAD Hercules, CA, USA) by
Analysis of eCBs and congeners comparing the position of relevant bands on the autora-
N-arachidonoylethanolamide (AEA), 2-arachidonoyl- diograms with those of the prestained standard mw or
monoacylglycerol (2-AG), palmitoylethanolamide (PEA) with those of the GAPDH bands, respectively. The ratio
and oleoylethanolamide (OEA) were extracted from tis- of the intensity of COX-2 bands to the intensity of
sues and plasma, purified and analysed as previously GAPDH bands was used to compare expression levels
described [43]. of these proteins following E.O. administration. One-
One-way ANOVA and the Bonferroni test for post way ANOVA and Fisher’s test for post hoc analyses
hoc analyses were applied to evaluate statistical differ- were applied to evaluate statistical differences among
ences among groups. groups.
Western blot Results
Tissue homogenates were prepared in 2% sodium dode- Fatty acid and eCB profiles in brain tissue
cyl sulphate (SDS). Protein concentrations were deter- Analysis of the frontal cortex (Figure 1A) and the
mined using the Lowry method of protein assay [44] remaining posterior cortex (Figure 1B) showed that, in
with bovine serum albumin as standard. Proteins for vehicle-treated rats, BCCAO/R affected fatty acid con-
each tissue homogenate (30 μg), diluted 1:1 in loading centration by causing about 10% decrease of DHA in
buffer, were heated to 95°C for 10 min and separated by
SDS-polyacrilamide gel electrophoresis (SDS-PAGE)
using a 12.5% (w/v) polyacrylamide resolving gel. Inter-
16 A
nal mw standards (Kaleidoscope Prestained Standards,
Bio-Rad, Hercules, CA, USA) were run in parallel. Two cids 15 a a
gels at a time were run for Coomassie staining and y a a
immunoblotting, respectively. Proteins for immunoblot- att
ting were electrophoretically transferred on a polyvinyli- al f 14 b
ot
dene fluoride membrane (Bio-Rad) using the Mini Trans of t 13
Blot Cell (Bio-Rad). Blots were blocked by immersion in %
20 mM Tris base and 137 mM sodium chloride (TBS)
12
containing 5% milk powder and 0.1% Tween 20 (TBS-
sham + vehicle sham + E.O. BCCAO/R + vehicle BCCAO/R + E.O.
T), for 60 min at room temperature and incubated over-
night at 4°C with rabbit polyclonal antibody against 16 B
COX-2 (residues 570-598) (Cayman Chem., USA), s
d
diluted 1:200 in TBS containing 5% milk powder and ci 15
a
0.02% sodium azide (NaN3) (Sigma-Aldrich, Steinheim, atty
Germany), was used as primary antiserum. After TBS-T al f 14
rinse, blots were incubated for 60 min, at room tem- ot
perature, with peroxidase-conjugated goat anti-rabbit of t 13
%
serum (Sigma Aldrich), diluted 1:10,000 in TBS/T.
Loading controls for equal loading of the wells were 12
obtained by stripping and immunostaining the mem- sham + vehicle sham + E.O. BCCAO/R + vehicle BCCAO/R + E.O.
branes as above, using a mouse monoclonal antibody
Figure1DHAconcentration,expressedasmol%oftotalfatty
against the housekeeping protein glyceraldehyde 3-phos- acids,infrontal(A)andposteriorcortex(B)ofratssubmitted
phate dehydrogenase (GAPDH) (Chemicon), diluted toshamoperationandtoBCCAO/R,pre-treatedeitherwiththe
1:1,000, as primary antiserum, and a peroxidase-conju- vehiclealoneorwiththeE.O..Errorbarsdepictstandard
deviation.Differentlettersdenotesignificantdifferences(p<0.05).
gated goat anti-mouse serum (Chemicon), diluted
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the frontal cortex (Figure 1A). No changes were other factors may intervene triggering a range of physio-
detected in the remaining posterior cortex (Figure 1B). logical responses to preserve structural lipid changes.
By contrast, no significant changes were evident in rats Previous studies reported failure of this model to
pre-treated with E.O. and submitted to BCCAO/R as achieve a reproducible ischemic insult in the rodent
compared with other groups (Figure 1). Analysis of brain due to the presence of efficient collateral systems,
eCBs and congeners revealed no changes either after which allow for a cerebral blood flow compensation
BCCAO/R or E.O. pre-treatment (data not shown). within a few minutes [45,46]. Results obtained show
that this model triggers a cerebral insult sufficient to
Fatty acid and eCB profiles in plasma cause a detectable, significant decrease in the tissue level
In order to verify whether there was a peripheral supply of DHA, the most abundant essential fatty acid of neu-
of DHA, we also measured levels of DHA and its pre- ronal membrane phospholipids [47]. DHA is particularly
cursor EPA in plasma. In rats pre-treated with E.O. susceptible to lipid peroxidation [48,49] and, for this
whether or not submitted to BCCAO/R, an increase in reason, potentially apt to contribute to hypoperfusion/
the DHA-to-EPA ratio (p < 0.05) was observed (Figure reperfusion-induced oxidative stress. However, several
2). Analysis of eCBs and congeners revealed that levels lines of evidence have associated increased levels of
of PEA and OEA increased significantly in rats pre-trea- DHA to tissue protection in neuroinflammation [50]
ted with E.O. and submitted to BCCAO/R (Figure 3) suggesting that DHA does not increase susceptibility to
compared to the vehicle-fed ones, whereas no change oxidative stress. Data in rodents and healthy humans
was observed in levels of AEA and 2-AG. support this inferring, as dietary supplementation with
n-3 PUFAs does not affect lipid peroxidation [51,52].
Western blot DHA is an agonist of peroxisome proliferator-activated
In the frontal cortex homogenate, the staining intensity receptor (PPAR) alpha [53]. Though mechanisms cer-
of the COX-2 protein band decreased significantly (p < tainly need further investigation, it can be suggested
0.05) after pre-treatment with E.O. in the rats submitted that PPARalpha, in turn, may act to attenuate the neu-
to BCCAO/R, as compared with the vehicle-fed rats roinflammatory reaction either by increasing the rate of
submitted to BCCAO/R (Figure 4). By contrast, no peroxisomal beta-oxidation or by slowing/suppressing
changes were observed between vehicle-fed rats, both nuclear factor-kappaB (NF-kB) activity and thereby
after BCCAO/R or after sham operation. slowing the activity of phospholipases and COX-2 [54].
The present findings demonstrate that just a single
Discussion dose of E.O. is sufficient to influence tissue metabolism
The present results show that BCCAO for 20 min fol- in response to BCCAO/R challenge. The BCCAO/R-
lowed by reperfusion for 30 min is apt to induce a weak induced decrease of DHA, most likely due to its break-
but reproducible insult which resulted in a decrease of down during lipid peroxidation, was totally prevented by
DHA and a parallel increase of COX-2 expression in the pre-treatment with E.O.. The observation that DHA/
frontal cortex, without any appreciable change in tissue EPA ratio raises after BCCAO/R following pre-treatment
morphology. This model has proved to be useful to test with E.O. may lead to suggest that there is an increased
E.O. effects in the early phase of BCCAO/R, before metabolism of n-3 which may supply damaged brain tis-
sue with newly produced DHA. On the other hand, the
increase of DHA/EPA ratio might be explained by an
augmented entry of EPA in the brain parenchyma to be
promptly metabolized to DHA [55,56]. Thus, in keeping
DHA/EPA
with the protecting role of DHA in neuroinflammation
4,0
[50], it is tempting to speculate that pre-treatment with
b
b E.O. affects the outcome of the BCCAO/R-induced
3,0 a a
events by modulating the tone of DHA precursors in
2,0 the brain. In this way, the sparing of DHA from lipid
peroxidation may contribute to minimize the extent of
1,0 the BCCAO/R-induced inflammatory reaction and to
help preserving the brain structure. The lower expres-
0,0
sham + vehicle sham + E.O. BCCAO/R + vehicle BCCAO/R + E.O. sion of COX-2 detected after BCCAO/R following pre-
Figure2DHA/EPAratioinplasmaofratssubmittedtosham treatment with E.O. compared to the treatment with the
operationandtoBCCAO/R,pre-treatedeitherwiththevehicle vehicle alone further supports this hypothesis. In fact,
aloneorwiththeE.O..Errorbarsdepictstandarddeviation. upregulation of COX-2 mRNA and protein, though not
Differentlettersdenotesignificantdifferences(p<0.05).
always predictive of harmful effects [57-60], is induced
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(cid:4)(cid:28)(cid:4) (cid:87)(cid:28)(cid:4)
(cid:1005)(cid:1006)(cid:1004) (cid:1005)(cid:1006)(cid:1004)
(cid:271)
(cid:373)(cid:258) (cid:1013)(cid:1004) (cid:373)(cid:258) (cid:1013)(cid:1004)
(cid:393)(cid:373)(cid:381)(cid:367)(cid:286)(cid:400)(cid:876)(cid:373)(cid:367)(cid:3)(cid:393)(cid:367)(cid:258)(cid:400) (cid:1007)(cid:1010)(cid:1004)(cid:1004) (cid:393)(cid:373)(cid:381)(cid:367)(cid:286)(cid:400)(cid:876)(cid:373)(cid:367)(cid:3)(cid:393)(cid:367)(cid:258)(cid:400) (cid:1007)(cid:1010)(cid:1004)(cid:1004) (cid:258) (cid:258) (cid:258)
(cid:1004) (cid:1004)
(cid:400)(cid:346)(cid:258)(cid:373)(cid:3)(cid:1085)(cid:3)(cid:448)(cid:286)(cid:346)(cid:349)(cid:272)(cid:367)(cid:286) (cid:400)(cid:346)(cid:258)(cid:373)(cid:3)(cid:1085)(cid:3)(cid:28)(cid:856)(cid:75)(cid:856) (cid:17)(cid:18)(cid:18)(cid:4)(cid:75)(cid:876)(cid:90)(cid:3)(cid:1085)(cid:3)(cid:448)(cid:286)(cid:346)(cid:349)(cid:272)(cid:367)(cid:286) (cid:17)(cid:18)(cid:18)(cid:4)(cid:75)(cid:876)(cid:90)(cid:3)(cid:1085)(cid:3)(cid:28)(cid:856)(cid:75)(cid:856) (cid:400)(cid:346)(cid:258)(cid:373)(cid:3)(cid:1085)(cid:3)(cid:448)(cid:286)(cid:346)(cid:349)(cid:272)(cid:367)(cid:286) (cid:400)(cid:346)(cid:258)(cid:373)(cid:3)(cid:1085)(cid:3)(cid:28)(cid:856)(cid:75)(cid:856) (cid:17)(cid:18)(cid:18)(cid:4)(cid:75)(cid:876)(cid:90)(cid:3)(cid:1085)(cid:3)(cid:448)(cid:286)(cid:346)(cid:349)(cid:272)(cid:367)(cid:286) (cid:17)(cid:18)(cid:18)(cid:4)(cid:75)(cid:876)(cid:90)(cid:3)(cid:1085)(cid:3)(cid:28)(cid:856)(cid:75)(cid:856)
(cid:1006)(cid:4)(cid:39) (cid:75)(cid:28)(cid:4)
(cid:1012)(cid:1004)(cid:1004) (cid:1005)(cid:1012)(cid:1004)
(cid:1011)(cid:1004)(cid:1004) (cid:1005)(cid:1009)(cid:1004) (cid:271)
(cid:373)(cid:258) (cid:1010)(cid:1004)(cid:1004) (cid:373)(cid:258)
(cid:373)(cid:367)(cid:3)(cid:393)(cid:367)(cid:258)(cid:400) (cid:1008)(cid:1009)(cid:1004)(cid:1004)(cid:1004)(cid:1004) (cid:373)(cid:367)(cid:3)(cid:393)(cid:367)(cid:258)(cid:400) (cid:1005)(cid:1013)(cid:1006)(cid:1004)(cid:1004) (cid:258)
(cid:381)(cid:367)(cid:286)(cid:400)(cid:876) (cid:1007)(cid:1004)(cid:1004) (cid:381)(cid:367)(cid:286)(cid:400)(cid:876) (cid:1010)(cid:1004) (cid:258) (cid:258)
(cid:393)(cid:373) (cid:1006)(cid:1004)(cid:1004) (cid:393)(cid:373)
(cid:1007)(cid:1004)
(cid:1005)(cid:1004)(cid:1004)
(cid:1004) (cid:1004)
(cid:400)(cid:346)(cid:258)(cid:373)(cid:3)(cid:1085)(cid:3)(cid:448)(cid:286)(cid:346)(cid:349)(cid:272)(cid:367)(cid:286) (cid:400)(cid:346)(cid:258)(cid:373)(cid:3)(cid:1085)(cid:3)(cid:28)(cid:856)(cid:75)(cid:856) (cid:17)(cid:18)(cid:18)(cid:4)(cid:75)(cid:876)(cid:90)(cid:3)(cid:1085)(cid:3)(cid:448)(cid:286)(cid:346)(cid:349)(cid:272)(cid:367)(cid:286) (cid:17)(cid:18)(cid:18)(cid:4)(cid:75)(cid:876)(cid:90)(cid:3)(cid:1085)(cid:3)(cid:28)(cid:856)(cid:75)(cid:856) (cid:400)(cid:346)(cid:258)(cid:373)(cid:3)(cid:1085)(cid:3)(cid:448)(cid:286)(cid:346)(cid:349)(cid:272)(cid:367)(cid:286) (cid:400)(cid:346)(cid:258)(cid:373)(cid:3)(cid:1085)(cid:3)(cid:28)(cid:856)(cid:75)(cid:856) (cid:17)(cid:18)(cid:18)(cid:4)(cid:75)(cid:876)(cid:90)(cid:3)(cid:1085)(cid:3)(cid:448)(cid:286)(cid:346)(cid:349)(cid:272)(cid:367)(cid:286) (cid:17)(cid:18)(cid:18)(cid:4)(cid:75)(cid:876)(cid:90)(cid:3)(cid:1085)(cid:3)(cid:28)(cid:856)(cid:75)(cid:856)
Figure3AEA,PEA,OEAand2-AGconcentrations,expressedaspmoles/ml,inplasmaofratssubmittedtoshamoperationandto
BCCAO/R,pre-treatedeitherwiththevehiclealoneorwiththeE.O..Errorbarsdepictstandarddeviation.Differentlettersdenotesignificant
differences(p<0.05).
by ischemia and plays a role in the ischemic brain injury remains to be elucidated. As it has been suggested that
[59,61-65]. Conversely, selective COX-2 inhibition ame- brain damage “spill over” effect may contribute to eCB
liorates brain damage and prevents neuronal death after and congeners plasma levels [16], it would be further
ischemia [66]. interesting to establish if E.O. may cause or modulate
An increase of eCB plasmatic levels has been shown in brain “spill over” in these conditions. On the other
several experimental models of ischemia [67]. In addi- hand, it has been widely demonstrated that PEA has
tion, in human stroke patients, an increase of PEA plas- important pharmacological effects in a wide range of
matic levels in early phases of ischemia has been experimental models of inflammation [68-70]. In parti-
demonstrated to be significantly correlated with neuro- cular, it is possible that PEA may exert neuroprotective
logical disability [16]. In the present study PEA and effects by preventing mast cell degranulation [71], acti-
OEA are increased only in BCCAO/R rats pre-treated vating PPARalpha [72] and dumping COX-2 expression
with E.O.. Whether and how E.O. administration may [68]. It could be speculated that the augmentation of
trigger an increase of PEA and OEA plasma levels peripheral PEA might represent the key factor for
Figure4(A)WesternblotanalysisofCOX-2inthefrontalcortexofratssubmittedtoshamoperationandtoBCCAO/R,pre-treated
eitherwiththevehiclealoneorwiththeE.O.(B)RelativelevelsofCOX-2expressionwithdensitometricanalysisofthegraylevelsexpressed
asapercentageoftheopticaldensity(O.D.)ratiooftheCOX-2-positivebandstotheGAPDH-positiveones.Errorbarsdepictstandarddeviation.
Differentlettersdenotesignificantdifferences(p<0.05).
Quartuetal.LipidsinHealthandDisease2012,11:8 Page8of10
http://www.lipidworld.com/content/11/1/8
enhancing PUFA metabolism via PPARalpha activation, Authordetails
protecting brain from ischemia/reperfusion injury. Inter- 1DepartmentofBiomedicalSciences,UniversityofCagliari,Cittadella
Universitaria,Monserrato,Italy.2DepartmentofChemicalSciences,University
estingly, exogenous administration of PEA in an acute ofCagliari,CittadellaUniversitaria,Monserrato,Italy.3Nutrisearchsrl,Pula,
stroke model is effective in reducing the infarct size Italy.
[73]. Moreover, in human stroke eCBs and congeners
Authors’contributions
become detectable in relation to the acute phase of
MQandSBconceivedthestudy,participatedinitsdesignandcoordination,
ischemic stroke [16] where they may play a role through draftandwrotethemanuscript;MPSconceivedandparticipatedinthe
multiple potential mechanisms [16]. designofthestudy,carriedouttheproteinanalysisandelaboratedthedata;
MBcarriedoutthetissuesamplingandperformedthestatisticalanalysisof
westernblotdata;GPandTMperformedthesurgery;LPhelpedinthe
Conclusions statisticalanalysis;MDFgavecriticalcontributiontothemanuscript;DF
performedtheSFE;GCandEM,participatedinthedesignofthestudy,
Previous studies tested the anti-oxidant, anti-microbial
performedtheeCBanalysisandelaboratedthedata;LCandAPperformed
and anti-inflammatory activities of the Pistacia lentiscus thefattyacidanalysisandelaboratedthedata.Allauthorsreadand
L. gum or fixed oil [25,27,28,32,33]. However, to our approvedthefinalmanuscript.
knowledge this is the first report on the effect of in vivo
Competinginterests
administration of Pistacia lentiscus L. essential oil. There- Theauthorsdeclarethattheyhavenocompetinginterests.
fore, identification of the E.O. constituent(s) that may
Received:24November2011 Accepted:12January2012
exert the beneficial effect in cerebral hypoperfusion/
Published:12January2012
reperfusion remains an issue worth further investigations.
A possible candidate is BCP, present in relatively high References
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