Modulation of the endocannabinoid system by focal brain
ischemia in the rat is involved in neuroprotection afforded
by 17b-estradiol
Diana Amantea
1
, Paola Spagnuolo
1,2
, Monica Bari
2,3
, Filomena Fezza
2,3
, Cinzia Mazzei
1
,
Cristina Tassorelli
4
, Luigi A. Morrone
1
, Maria T. Corasaniti
3,5
, Mauro Maccarrone
3,6,
* and
Giacinto Bagetta
1,
*
1 Department of Pharmacobiology and University Center for the Study of Adaptive Disorder and Headache (UCADH),
Section of Neuropharmacology of Normal and Pathological Neuronal Plasticity, University of Calabria, Rende (CS), Italy
2 Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Rome, Italy
3 IRCCS Neurological Institute C. Mondino Foundation, Mondino-Tor Vergata Center for Experimental Neuropharmacology,
Laboratory of Neurochemistry, Rome, Italy

artery occlusion were further potentiated by 1 h of reperfusion, whereas
anandamide binding to type 1 cannabinoid and type 1 vanilloid receptors
was not affected significantly by the ischemic insult. Additionally, the can-
nabinoid type 1 receptor antagonist SR141716, but not the receptor agonist
R-(+)-WIN55,212-2, significantly reduced (33%; P < 0.05) cerebral infarct
volume detected 22 h after the beginning of reperfusion. A neuroprotective
intraperitoneal dose of 17b-estradiol (0.20 mgÆkg
)1
) that reduced infarct
size by 43% also minimized the effect of brain ischemia on the endocanna-
binoid system, in an estrogen receptor-dependent manner. In conclusion,
we show that the endocannabinoid system is implicated in the pathophysi-
ology of transient middle cerebral artery occlusion-induced brain damage,
and that neuroprotection afforded by estrogen is coincident with a re-
establishment of anandamide levels in the ischemic striatum through a
mechanism that needs to be investigated further.
Abbreviations
AEA, anandamide (arachidonoylethanolamide); CB, cannabinoid; CNS, central nervous system; E
2
,17b-estradiol; ER, estrogen receptor;
FAAH, fatty acid amide hydrolase; MCA, middle cerebral artery; MCAo, middle cerebral artery occlusion; NAPE, N-acylphosphatidyl-
ethanolamine; NAPE-PLD,N-acylphosphatidylethanolamine-hydrolyzing phospholipase D; NArPE, N-arachidonoylphosphatidylethanolamine;
RTX, resinferatoxin; TRPV1, transient receptor potential vanilloid-1; TTC, 2,3,5-triphenyltetrazolium chloride.
4464 FEBS Journal 274 (2007) 4464–4475 ª 2007 The Authors Journal compilation ª 2007 FEBS
Endocannabinoids are amides, esters and ethers of
long-chain polyunsaturated fatty acids that are synthe-
sized on demand. Anandamide (arachidonoylethanol-
amide) (AEA) was the first member of this family to
be discovered [1], and it is synthesized by the enzyme
N-acylphosphatidylethanolamine (NAPE)-hydrolyzing

gested to represent an endogenous protective mecha-
nism during CNS injury [16]. By contrast, recent
studies have suggested that endogenously released en-
docannabinoids may be toxic to neurons in animal
models of acute neurodegeneration. Thus, for instance,
both CB1 receptor stimulation and blockade have been
shown to exert neuroprotection in rodent models of
focal brain ischemia [14,15,17].
Recent studies have highlighted the ability of estro-
gens to enhance recovery from ischemic brain injury
resulting from cardiovascular disease or cerebrovascu-
lar stroke. 17b-estradiol (E
2
) has been shown to reduce
mortality and cerebral damage in a variety of animal
models of acute cerebral ischemia, including transient
and permanent MCAo [18–20], photothrombotic focal
ischemic brain damage [21], and global forebrain ische-
mia [22,23]. Accordingly, administration of either
pharmacologic or physiologic doses of E
2
provides
neuroprotection in ovariectomized female rodents
subjected to focal brain ischemia [18–20,24]. Similar
results have been obtained in male rats, as either acute
or chronic E
2
administration significantly reduces brain
damage following transient MCAo [25].
Although the neuroprotective effects of E

tors, mediating rapid, nongenomic actions, or recep-
tor-independent mechanisms, mainly due to the
antioxidant free radical-scavenging properties of the
steroidal molecules [35]. However, the exact contribu-
tion of each molecular mechanism to the overall neu-
rotrophic and neuroprotective effect of estrogens is
still a matter of debate.
Interestingly, recent studies have revealed that sex
hormones may provide pivotal modulation of the
endocannabinoid system in a tissue- and species-spe-
cific manner, as demonstrated both in vivo, in mouse
uterus, and in vitro, in human endothelial, lymphoma
and neuroblastoma cells [36,37]. In particular, the
endocannabinoid AEA is released from human endo-
thelial cells treated with E
2
, and complements some
actions of this hormone on human platelets [38].
However, the modulation of the endocannabinoid
system by estrogen in the brain has been poorly
investigated.
In the present study, we aimed to evaluate the effect
of MCAo-induced brain insult on AEA regional level,
metabolism, and receptor binding and expression.
The putative neuroprotective action of agonists and
antagonists of cannabinoid receptors has also been
investigated. Moreover, we demonstrate here that
D. Amantea et al. Endocannabinoid system modulation by E
2
after MCAo

NAPE-PLD activity in the cerebral cortex, and this is
consistent with the lack of significant changes in
endogenous AEA levels detected in this ischemic corti-
cal region (Fig. 1A).
The increase in endogenous AEA levels detected in
the striatum was persistent also at later stages of reper-
fusion following 2 h of MCAo (Fig. 2). By contrast,
cortical levels of AEA, which did not significantly
change after 1 h of reperfusion, were significantly
reduced 6 h or 22 h later (Fig. 2).
Unlike endocannabinoid metabolism, which appears
to be modified as a consequence of focal brain ische-
mia, CB1 and TRPV1 receptor binding in cortices and
striata did not change following 2 h of MCAo, either
in the absence or in the presence of 1 h of reperfusion
(data not shown).
Fig. 1. Endogenous levels of AEA (A) and activity of FAAH (B) and
NAPE-PLD (C) in the ischemic striatum and cortex of rats subjected
to 2 h of MCAo, with or without 1 h of reperfusion. Sham rats
were exposed to the same surgical procedure without occlusion of
the MCA. E
2
(0.20 mgÆkg
)1
, intraperitoneal) was administered 1 h
before MCAo. Values are expressed as mean ± SD (n ¼ 3), and
were analyzed by the Mann–Whitney U-test. *P < 0.01 versus
Sham;
#
P < 0.01 versus MCAo;

the cannabinoid receptor agonist R-(+)-WIN-55,212-2
(1 mgÆkg
)1
, intraperitoneal, 15 min before MCAo) did
not affect brain infarct damage produced by transient
MCAo (Fig. 4D).
Estrogens are known to protect the brain against
focal ischemia [35]. In order to investigate the role of
the endocannabinoid system in the neuroprotection
afforded by estrogen, the effect of acute treatment with
E
2
on endogenous AEA levels in both ischemic cortex
and striatum was evaluated. The results showed that
E
2
(0.20 mgÆkg
)1
, intraperitoneal) administered 1 h
before MCAo significantly reversed the increase of
endogenous AEA levels produced by 2 h of focal cere-
bral ischemia in the striatum (Fig. 1A). Moreover,
FAAH and NAPE-PLD activities returned to basal
(sham) levels when rats were treated with the same
dose of E
2
1 h prior to MCAo (Fig. 1B,C). It seems of
further interest that, although brain ischemia did not
alter cannabinoid receptor expression, E
2

)1
, intraperitoneal) was administered 1 h
before MCAo. Values are expressed as mean ± SD (n ¼ 4), and
were analyzed by the Mann–Whitney U-test. **P < 0.05 versus
Sham;
§
P < 0.05 versus MCAo.
1.0 6.0 22.0
0
50
100
150
200
Striatum Cortex
**
*
***
*
,#
*
,##
0
Reperfusion (h)
Endogenous levels of AEA
(% of control)
Fig. 2. Endogenous levels of AEA in the striatum and cortex of rats
subjected to 2 h of MCAo, followed by 0, 1, 6 or 22 h of reperfu-
sion (100% as MCAo samples in Fig. 1A). Values are expressed as
mean ± SD (n ¼ 3), and were analyzed by the Mann–Whitney
U-test. *P < 0.05, **P < 0.01 and ***P < 0.001 versus 0 h of

volume produced by 2 h of MCAo followed by
22 h of reperfusion. The neuroprotection afforded by
E
2
was reverted by the ER antagonist ICI182 780
(0.25 mgÆkg
)1
, intraperitoneal), administered 1 h prior
to E
2
(Fig. 6).
Discussion
The results reported in the present study demonstrate
that a focal ischemic brain insult produced by transient
MCAo results in a significant increase of endogenous
AEA levels in the ischemic striatum, as early as 2 h
following injury. This effect was associated with
altered endocannabinoid metabolism, as 2 h of MCAo
also resulted in reduced activity and expression of the
metabolic enzyme FAAH, whereas NAPE-PLD activ-
ity was significantly increased. Interestingly, we
observed that reperfusion increased striatal AEA levels
above those detected after 2 h of MCAo, thus suggest-
ing that re-establishment of blood supply may further
Fig. 4. SR141716, a selective CB1 receptor antagonist, but not WIN55,212-2, a CB1 receptor agonist, reduces brain infarct size following
transient MCAo. The right MCA was occluded for 2 h with a nylon suture, as described in Experimental procedures, and cerebral infarct vol-
ume was evaluated 22 h after reperfusion. Eight serial sections from each brain were cut at 2 mm intervals from the frontal pole and incu-
bated in TTC, which stains viable tissue red but not infarcted areas (C). The infarct volume was calculated by summing the infarcted area of
the eight sections (A) and multiplying by the interval thickness between sections. Rats received vehicle (vegetable oil, n ¼ 5) or SR141716
(3 mgÆkg

NAPE-PLD activity [pmolÆmin
)1
Æ(mg protein)
)1
] 22±3 25±3
CB1 receptor binding [fmolÆ(mg protein)
)1
] 190 ± 20 180 ± 20
Endocannabinoid system modulation by E
2
after MCAo D. Amantea et al.
4468 FEBS Journal 274 (2007) 4464–4475 ª 2007 The Authors Journal compilation ª 2007 FEBS
contribute to endocannabinoid modulation. The latter
hypothesis is supported by the evidence that the
increase in NAPE-PLD activity was more pronounced
following 1 h of reperfusion, as compared to the enzy-
matic activity measured after MCAo alone. Thus, it is
conceivable that an early increase in endogenous AEA
levels in the ischemic striatum, which comprises most
of the ischemic core [39], might underlie brain damage
produced by focal ischemia. This effect appears to
occur via activation of cannabinoid receptors, as pre-
treatment with the CB1 receptor antagonist SR141716
afforded neuroprotection in rats subjected to transient
MCAo.
An early increase of AEA has been previously
reported in the whole brain of rats following transient
focal brain ischemia [15]. However, in that study, no
information was collected about the alterations
induced by the ischemic insult in different brain

P < 0.01 versus
MCAo;
§
P < 0.05 versus MCAo;
@
P < 0.01 versus MCAo + E
2
;
&
P < 0.05 versus MCAo + E
2
.
Vehicle E
2
ICI + E
2
0
200
400
600
**
Infarct volume (mm
3
)
0 1 2 3 4 5 6 7 8
0
25
50
75
B

2
). Values are expressed as mean ± SEM (n ¼ 5), and were ana-
lyzed by
ANOVA followed by Tukey’s post hoc test. **P < 0.01 ver-
sus vehicle.
D. Amantea et al. Endocannabinoid system modulation by E
2
after MCAo
FEBS Journal 274 (2007) 4464–4475 ª 2007 The Authors Journal compilation ª 2007 FEBS 4469
suggested to represent an endogenous protective mecha-
nism during CNS injury [16]. In line with this, exo-
genously administered (endo)cannabinoids have been
shown to protect neurons via several mechanisms, yet
the role of endogenously released endocannabinoids on
neuronal damage appears to be controversial [9]. In
fact, recent studies have paradoxically suggested that
endogenously released endocannabinoids may be toxic
to neurons in animal models of acute neurodegenera-
tion. Accordingly, administration of the CB1 receptor
antagonist SR141716 evoked a significant neuroprotec-
tive response in adult rats subjected to permanent or
transient MCAo [14,15], and in neonatal rats exposed
to an intrastriatal microinjection of NMDA [41]. This is
consistent with our data, documenting that systemic
administration of SR141716 results in a significant
reduction of brain infarct volume produced by transient
MCAo, thus suggesting that increased AEA levels pro-
duced during the early stages of brain ischemic insult
may trigger neurodegenerative events through activa-
tion of CB1 receptors. It seems noteworthy that, despite

accumulation of the corresponding precursors
NAPE [11,14]. Here, we found that the activity of the
AEA-synthesizing enzyme NAPE-PLD was signifi-
cantly increased following MCAo, and that this was
paralleled by a significant reduction in the activity and
expression of the AEA-hydrolyzing enzyme FAAH.
Therefore, our data suggest that accumulation of endog-
enous AEA during focal ischemic injury may stem from
a specific mechanism involving altered endocannabinoid
metabolism.
To the best of our knowledge, there is no informa-
tion on the putative modulation of the endocannabi-
noid system by E
2
in the brain under pathophysiologic
conditions. Here, we show that acute administration of
a pharmacologic dose of E
2
to male rats prevents the
increase in AEA levels produced in the striatum by
MCAo, an effect that seems to occur through the
modulation of both NAPE-PLD and FAAH. In fact,
both enzyme activities returned to control values when
rats were pretreated with a neuroprotective dose of the
hormone. It seems also noteworthy that E
2
reduced
CB1 receptor binding in the ischemic striatum, and it
is tempting to speculate that this may further contrib-
ute to neuroprotection by reducing the ability of

pathologic conditions, leading to the suggestion that it
might be pivotal in hormone-mediated neuroprotection
after ischemic stroke.
Endocannabinoid system modulation by E
2
after MCAo D. Amantea et al.
4470 FEBS Journal 274 (2007) 4464–4475 ª 2007 The Authors Journal compilation ª 2007 FEBS
Experimental procedures
Materials
Chemicals were of the purest analytical grade. AEA,
resinferatoxin (RTX), E
2
and R-(+)-WIN55,212-2 were
obtained from Sigma Chemical Co. (St Louis, MO).
ICI182 780 was purchased from Tocris Bioscience
(Avonmouth, UK). [
3
H]AEA (223 CiÆmmol
)1
), [
3
H]RTX
(43 CiÆmmol
)1
) and [
3
H]CP55.940 (5-(1,1¢-dimethylheptyl)-
2-[1R,5R-hydroxy-2R-(3-hydroxypropyl) cyclohexyl]-phenol,
126 Ci mmol
)1

)1
,
1 h before E
2
. SR141716 was dissolved in vegetable oil and
administered intraperitoneally at a dose of 3 mgÆkg
)1
,
15 min prior to MCAo. R-(+)-WIN55,212-2 was dissolved
in propylene glycol and administered intraperitoneally at a
dose of 1 mgÆkg
)1
, 15 min prior to MCAo. Control rats
received a vehicle in which the corresponding drug had
been dissolved and that was administered under the same
injection schedule as the drug treatment.
All the experimental procedures were performed in accor-
dance with the guidelines of the European Community
Council Directive 86⁄ 609, included in D.M. 116 ⁄ 1992 of
the Italian Ministry of Health.
Focal cerebral ischemia
Brain ischemia was induced by MCAo in male Wistar rats
(280–320 g) by intraluminal filament, using the relatively
noninvasive technique previously described by Longa et al.
[58]. Briefly, rats were anesthetized with 5% isoflurane in
air, and were maintained with the lowest acceptable concen-
tration of the anesthetic (1.5–2%). Body temperature was
measured with a rectal probe and was kept at 37 °C during
the surgical procedure with a heating pad. Under an oper-
ating microscope, the external and internal right carotid

decapitation, and the brains were rapidly removed. Eight
serial sections from each brain were cut at 2 mm intervals
from the frontal pole using a rat brain matrix. To measure
ischemic damage, brain slices were stained in a solution
containing 2% 2,3,5-triphenyltetrazolium chloride (TTC) in
saline, at 37 °C. After 10 min of incubation, the slices were
transferred to 10% neutral buffered formaldehyde and
stored at 4 °C prior to analysis. Images of TTC-stained sec-
tions were captured using a digital scanner and analyzed
using image analysis software (imagej, version 1.30). The
infarct volume (mm
3
) was calculated by summing the
infarcted area (unstained) of the eight sections and multi-
plying by the interval thickness between sections [60].
Analysis of the endocannabinoid system
For analysis of the endocannabionoid system, rats were
killed by decapitation at different times following MCAo,
as indicated; the brains were rapidly dissected out, and
D. Amantea et al. Endocannabinoid system modulation by E
2
after MCAo
FEBS Journal 274 (2007) 4464–4475 ª 2007 The Authors Journal compilation ª 2007 FEBS 4471
ipsilateral cortical and striatal samples were frozen in liquid
nitrogen.
For the evaluation of endogenous levels of AEA, rat
brain samples were homogenized with an UltraTurrax
T25 (Stauffen, Germany) in 50 mm Tris ⁄ HCl, 1 mm
EDTA (pH 7.4) and 1 mm phenylmethanesulfonyl fluoride
buffer, at a 1 : 10 (w ⁄ v) homogenization ratio. Lipids

. The synthesis of AEA through
the activity of NAPE-PLD (EC 3.1.4.4) was assayed in brain
homogenates (50 lg per test
1
), using 100 lm N-[
3
H]Arachi-
donoyl-phosphatidylethanolamine, as previously reported
[64]. NAPE-PLD activity was expressed as pmol AEA
releasedÆmin
)1
Æ(mg protein)
)1
. It should be mentioned that a
novel biosynthetic pathway for AEA has been recently
reported in mouse brain and RAW264.7 macrophages [65].
This pathway involves the phospholipase C-catalyzed cleav-
age of NAPE to generate a phosphoanandamide, which is
subsequently dephosphorylated by phosphatases. Therefore,
NAPE hydrolysis assayed in this study may not be the only
mechanism responsible for the production of AEA. The
binding of 400 pm [
3
H]CP55.940 to rat brain membranes was
determined through rapid filtration assays [63], and was
expressed as fmol CP55.940 boundÆ(mg protein)
)1
. Also, the
binding of 200 pm [
3

della Salute (RC 2005), Istituto Superiore di Sanita
`
(AIDS Project 2005), MIUR (PRIN 2004, prot.
2004053099-004) and Fondazione della Cassa di
Risparmio di Teramo (TERCAS 2004) is also grate-
fully acknowledged.
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