Báo cáo khoa học: Modulation of the endocannabinoid system by focal brain ischemia in the rat is involved in neuroprotection afforded by 17b-estradiol pdf

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

4 Laboratory of Pathophysiology of Integrative Autonomic Systems, IRCCS Neurological Institute C. Mondino Foundation and University
Centre for the Study of Adaptive Disorder and Headache (UCADH), Pavia, Italy
5 Department of Pharmacobiological Sciences, University Magna Graecia of Catanzaro, Italy
6 Department of Biomedical Sciences, University of Teramo, Italy
Keywords
endocannabinoids; estrogen; middle cerebral
artery occlusion; stroke
Correspondence
G. Bagetta, Department of Pharmacobiology,
University of Calabria, via P. Bucci Ed.
Polifunzionale, 87036 Rende (CS), Italy
Fax: +39 0984 493462
Tel: +39 0984 493462
E-mail:
*These authors contributed equally to this
work
(Received 28 March 2007, revised 18 June
2007, accepted 3 July 2007)
doi:10.1111/j.1742-4658.2007.05975.x
Endogenous levels of the endocannabinoid anandamide, and the activities
of the synthesizing and hydrolyzing enzymes, i.e. N-acylphosphatidyletha-
nolamine-hydrolyzing phospholipase D and fatty acid amide hydrolase,
respectively, were determined in the cortex and the striatum of rats sub-
jected to transient middle cerebral artery occlusion. Anandamide content
was markedly increased ( 3-fold over controls; P < 0.01) in the ischemic
striatum after 2 h of middle cerebral artery occlusion, but not in the cortex,
and this elevation was paralleled by increased activity of N-acylphosphati-
dylethanolamine-hydrolyzing phospholipase D ( 1.7-fold; P < 0.01),
and reduced activity ( 0.6-fold; P < 0.01) and expression ( 0.7-fold;
P < 0.05) of fatty acid amide hydrolase. These effects of middle cerebral

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

phospholipase D (NAPE-PLD) [2]. Following cellular
depolarization and Ca
2+
influx, endocannabinoids are
released into the extracellular space and interact with
type 1 and type 2 cannabinoid (CB1 and CB2) recep-
tors, non-CB1 ⁄ non-CB2 receptors, and noncannabi-
noid receptors, including the type 1 vanilloid receptor
[transient receptor potential vanilloid-1 (TRPV1)], a
ligand-gated and nonselective cationic channel [3]. The
biological actions of AEA cease following cellular
uptake, mediated by a membrane transporter [4], and
subsequent intracellular degradation catalyzed by a
fatty acid amide hydrolase (FAAH), which cleaves the
amide bond to form arachidonic acid and ethanol-
amine [5]. Taken together, AEA, its congeners and the
proteins that bind, synthesize or transport them form
the ‘endocannabinoid system’ [6].
In the brain, endocannabinoids act as retrograde
messengers to control multiple central nervous system
(CNS) functions, including learning and memory, pain,
sleep, and appetite [7]. Moreover, there is experimental
evidence to support a dual role for AEA in the CNS
as a neuroprotective or neurotoxic agent [8,9]. Endo-
cannabinoids are indeed elevated in a variety of acute
neurodegenerative insults, such as decapitation-induced
ischemia [10], N-methyl-d-aspartate (NMDA)-induced
excitotoxicity [11], convulsions [12], traumatic brain
injury [13], and notably middle cerebral artery (MCA)
occlusion (MCAo) [14,15]. This elevation has been sug-

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

2
in
humans are controversial [26], there is evidence that E
2
enhances recovery from brain injury following cerebral
ischemia [27,28], and continued use of estrogens has
been shown to significantly reduce the risk of stroke
[29–31]. This is also confirmed by epidemiologic evi-
dence indicating that women are more protected than
men against stroke until the menopause [27]. However,
recent large, randomized, clinical trials have questioned
the effectiveness of female sex hormones in the preven-
tion of coronary heart disease and stroke [32–34].
Several mechanisms have been suggested to underlie
E
2
neuroprotection, including modulation of synapto-
genesis, protection against apoptosis, anti-inflamma-
tory activity, and increased cerebral blood flow.
Estrogens exert their activity through the interaction
with intracellular estrogen receptors (ERs), ERa and
ERb, which results in the modulation of the transcrip-
tion of estrogen target genes, including those impli-
cated in neuronal survival. ER activation may also
mediate rapid nongenomic effects of E
2
via interaction
with intracellular signaling cascades. However, there is
evidence documenting that neuroprotection may also
occur via interaction with ER-like membrane recep-

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

FEBS Journal 274 (2007) 4464–4475 ª 2007 The Authors Journal compilation ª 2007 FEBS 4465
modulation of the endocannabinoid system is implicated
in the mechanisms of neuroprotection afforded by acute
administration of a pharmacologic dose of estrogen in
male rats.
Results
Two hours of MCAo resulted in a significant increase in
endogenous AEA levels in the striatum ipsilateral to the
ischemic damage, but not in the cerebral cortex. Inter-
estingly, when reperfusion was allowed for 1 h following
2 h of MCAo, endogenous levels of AEA were higher
than those detected in the striata of rats subjected to
brain ischemia without reperfusion (Fig. 1A).
In order to evaluate whether ischemia-induced changes
in endogenous AEA levels were associated with altered
endocannabinoid metabolism, the activity of FAAH was
measured in cortices and striata from rats with focal
brain ischemia. Two hours of MCAo, with or without
1 h of reperfusion, resulted in a significant decrease in
FAAH activity as detected in the striatum, but not in the
cortex, ipsilateral to the ischemic damage (Fig. 1B).
Furthermore, increased AEA levels in the ischemic
striatum were also associated with a significant
increase in NAPE-PLD activity, as detected following
2 h of MCAo (Fig. 1C). More interestingly, re-estab-
lishment of the blood supply for 1 h resulted in a more
pronounced increase in the activity of NAPE-PLD, as
compared to the enzymatic activity measured in striata
after 2 h of MCAo without reperfusion (Fig. 1C). By
contrast, focal brain ischemia did not appear to affect

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;

§
P < 0.05 versus MCAo.
Endocannabinoid system modulation by E
2
after MCAo D. Amantea et al.
4466 FEBS Journal 274 (2007) 4464–4475 ª 2007 The Authors Journal compilation ª 2007 FEBS
The lack of change in CB1 receptor binding capacity
following MCAo was also confirmed by data showing
that CB1 receptor striatal content was not modified by
focal ischemic insult (Fig. 3). By contrast, striatal con-
tent of the metabolic enzyme FAAH was significantly
reduced following 2 h of MCAo, with or without 1 h
of reperfusion (Fig. 3). The latter finding is consistent
with the reduced activity of FAAH in the ischemic stri-
atum of rats that have undergone MCAo (Fig. 1B).
The lack of specific antibodies to NAPE-PLD pre-
vented us from further extending the analysis of pro-
tein content to this enzyme.
In order to evaluate whether increased AEA levels
following MCAo might contribute to ischemic brain
damage or, conversely, might serve as an endogenous
neuroprotective mechanism, we assessed the effect of
CB1 receptor blockade or activation on ischemic dam-
age. We found that administration of the CB1 recep-
tor antagonist SR141716 (3 mgÆkg
)1
, intraperitoneal),
15 min before MCAo, resulted in a significant reduc-
tion in brain infarct volume as detected 22 h after rep-
erfusion (Fig. 4A–C). By contrast, pretreatment with

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

pretreatment
resulted in a significant (45%) reduction of CB1 bind-
ing in the striatum, but not in the cortex ipsilateral to
the ischemic insult (data not shown). Instead, CB1
receptor content was not affected by the hormone
treatment (Fig. 3), and neither was TRPV1 binding
(data not shown).
Interestingly, E
2
does not appear to significantly
modulate basal levels of AEA, FAAH and NAPE-
PLD activity and CB1 receptor binding as assessed in
striatal samples from sham-operated rats, pretreated
with E
2
or vehicle, 3 h before sacrifice (Table 1). This
suggests that neuropathologic alterations of the endoc-
annabinoid system, such those detected after MCAo,
are instrumental for its modulation by estrogen.
The modulation of the endocannabinoid system by
E
2
in the ischemic striatum seems to involve the activa-
tion of intracellular ERs. In fact, administration of the
Fig. 3. FAAH and CB1 receptor content in the striatum of rats sub-
jected 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 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

reperfusion;
#
P < 0.01 and
##
P < 0.001 versus 1 h of reperfusion.
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 4467
ER antagonist ICI182 780 (0.25 mgÆkg
)1
, intraperito-
neal, 1 h prior to E
2
) was able to significantly antago-
nize the effects of E
2
(0.20 mgÆkg
)1
, intraperitoneal,
1 h before MCAo) on endogenous levels of AEA, on
FAAH and NAPE-PLD activity, and on CB1 receptor
binding in the striatum (Fig. 5).
Interestingly, acute treatment with E
2
(0.20 mgÆkg
)1
,
intraperitoneal), given 1 h before the ischemic insult,
resulted in a significant reduction of brain infarct

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

)1
, n ¼ 4) intraperitoneally, 15 min prior to MCAo (A–C). In another set of experiments, rats received vehicle (propylene glycol,
n ¼ 7) or WIN55,212-2 (1 mg kg
)1
, n ¼ 7) intraperitoneally, 15 min prior to MCAo (D).Values are expressed as mean ± SEM, and were
compared by unpaired two-tailed t-test. *P < 0.05 versus vehicle.
Table 1. Effect of acute administration of E
2
on the endocannabi-
noid system in striatal tissue from sham-operated rats. Rats were
treated with E
2
(0.2 mgÆkg
)1
, intraperitoneal) or vehicle (vegetable
oil, 1 mLÆkg
)1
, intraperitoneal), 3 h before sham operation. Values
are expressed as mean ± SD (n ¼ 3), and were analyzed by the
Mann–Whitney U-test.
Vehicle E
2
Endogenous AEA [pmolÆ(mg protein)
)1
] 35±3 30±9
FAAH activity [pmolÆmin
)1
Æ(mg protein)
)1
] 820 ± 80 884 ± 90

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

regions, and neither was the biochemical background
behind the effect of MCAo on AEA levels investigated
[15]. We did observe an early significant increase in
endogenous AEA levels in the ischemic striatum but
not in the cortex of rats subjected to MCAo. The lack
of acute changes in endocannabinoid levels in the
cortical regions may stem from differential regional
susceptibility to the ischemic insult, 2 h of MCAo
being not enough to produce significant AEA elevation
in the penumbral region. By contrast, we did observe a
reduction in AEA levels in the cortex at later stages of
reperfusion, which may indeed be the result of delayed
damage, as compared to the striatum [39]. However,
the exact pathophysiologic significance of the latter
observation needs to be investigated further.
Endogenous levels of AEA are elevated by decapita-
tion-induced ischemia [10], NMDA-induced excitotox-
icity in vivo [11], neonatal traumatic brain injury [11],
kainate-induced neuronal excitation [40] and, most
notably, MCAo [15]. This elevation of AEA has been
Fig. 5. The observed effects of E
2
on endogenous levels of AEA,
on FAAH and NAPE-PLD activity, and on CB1 receptor binding in
the striatum of rats following MCAo appear to be mediated by E
2
receptor stimulation, as these effects are reversed by ICI182 780,
a pure ER antagonist. Values are expressed as mean ± SD (n ¼ 4),
and analyzed by the Mann–Whitney U-test.
#

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

A
Vehicle
E
2
ICI + E
2
coronal section
Infarct area (mm
2
)
Fig. 6. Neuroprotection afforded by E
2
against brain damage pro-
duced by transient MCAo is reversed by ICI182 780, a pure ER
antagonist. The right MCA was occluded for 2 h with a nylon
suture, as described in Experimental procedures, and cerebral
infarct volume was evaluated 22 h after reperfusion. Eight serial
sections from each brain were cut at 2 mm intervals from the fron-
tal pole and incubated in TTC, which stains viable tissue red but
not infarcted areas. The infarct volume (B) was calculated by sum-
ming the infarcted area of the eight sections (A) and multiplying by
the interval thickness between sections. Rats received E
2
(0.20 mgÆkg
)1
, intraperitoneal, 1 h before MCAo), alone or in combi-
nation with ICI182 780 (0.25 mgÆkg
)1
, intraperitoneal, 1 h prior to
E

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

the acute neuronal injury that occurs in the ischemic
striatum following MCAo, under the present experi-
mental conditions CB1 receptor expression and ligand-
binding capacity are not compromised. CB1 receptors
are predominantly localized on presynaptic nerve termi-
nals, and their stimulation can elicit either inhibitory
effects by blocking glutamate release or excitatory
effects by blocking 4-aminobutyric acid (GABA)
release, depending on which neuronal circuits are acti-
vated [7,42]. Although inhibition of glutamate release
has been suggested to represent a pivotal mechanism
involved in endocannabinoid-mediated neuroprotection
[17,43–46], CB1 receptor-induced reduction of the
inhibitory GABAergic input in the striatum [47] may
conversely provide a mechanism underling neurodegen-
eration. Moreover, activation of CB1 receptors local-
ized on cerebral blood vessels has been suggested to
determine altered autoregulation of cerebral blood flow
[48–50], and this may further contribute to brain dam-
age following the ischemic insult. Thus, although it can-
not be excluded that AEA may be neurotoxic via
activation of molecular targets distinct from CB1, our
data suggest that neurotoxicity occurs through CB1
receptor activation. Accordingly, cannabinoid receptor
activation may induce [51] or prevent [52] apoptosis,
implying that CB1 receptors represent a key regulator
of cell survival ⁄ death and a useful pharmacologic target
to control cell death in neurodegenerative diseases.
Increased levels of N-acylethanolamines following
brain injury have been suggested to depend on

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

endogenous cannabinoids to evoke CB1-mediated
responses. Moreover, we report the original observa-
tion that E
2
increased FAAH and reduced NAPE-
PLD activity via an ER-dependent mechanism in the
ischemic striatum, thus reversing the effects of ischemia
on these enzymatic activities. Transient MCAo has
been associated with blood–brain barrier disruption
[54,55], and under these experimental conditions the
antiestrogen ICI182 780 has been shown to reach the
brain after systemic administration [56,57]. Thus, it is
plausible that the drug is able to cross the blood–brain
barrier under our experimental conditions.
Collectively, our study demonstrates that focal brain
ischemia produced by transient MCAo results in a sig-
nificant modulation of the endocannabinoid system,
which occurs as early as 2 h following injury and con-
tinues during the early stages of reperfusion in the
ischemic striatum. Striatal downregulation of FAAH
and upregulation of NAPE-PLD activity lead to
increased levels of AEA, which in turn may play a role
in the pathophysiology of damage occurring in the
ischemic brain. More interestingly, we found that the
putative neurotoxic effects produced by the MCAo-
induced increase of endogenous AEA levels may be
significantly blocked by estrogen, possibly through an
ER-dependent mechanism. In conclusion, this is the
first report documenting the modulation of the endo-
cannabinoid system by estrogen in the brain under

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

) were purchased from Perkin Elmer Life
Sciences (Boston, MA). N-[
3
H]Arachidonoyl-phosphatidyl-
ethanolamine (200 CiÆmmol
)1
) was obtained from ARC
(St Louis, MO). N-piperidino-5-(4-chlorophenyl)-1-(2,4-di-
chlorophenyl)-4-methyl-3-pyrazole carboxamide (SR141716)
was a kind gift of Sanofi-Aventis Recherche (Montpellier,
France). Rabbit polyclonal antibodies to CB1R were
obtained from Cayman Chemicals (Ann Arbor, MI), rabbit
polyclonal antibodies to FAAH [53] were prepared by
Primm S.r.l. (Milan, Italy), and goat anti-(rabbit alkaline
phosphatase) conjugates (GAR-AP) were obtained from
Bio-Rad Laboratories (Hercules, CA).
Animals and drug treatments
Adult male Wistar rats were purchased from Charles River,
Calco, Italy. Animals were housed under controlled envi-
ronmental conditions with an ambient temperature of
22 °C, a relative humidity of 65%, and a 12 h light : 12 h
dark cycle, with free access to food and water. E
2
was dis-
solved in vegetable oil and administered intraperitoneally,
1 h prior to MCAo, at a dose of 0.20 mgÆkg
)1
. ICI182 780
was dissolved in 4% dimethylsulfoxide in vegetable oil and
administered intraperitoneally at a dose of 0.25 mgÆkg

)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

arteries were exposed through a neck incision. The external
carotid artery was cut approximately 3 mm above the com-
mon carotid artery bifurcation, and a silk suture was tied
loosely around the external carotid stump. A silicone-
coated nylon filament (diameter: 0.28 mm) was then
inserted into the external carotid artery and gently
advanced into the internal carotid artery, approximately
18 mm from the carotid bifurcation, until mild resistance
was felt, thereby indicating occlusion of the origin of the
MCA in the Willis circle. The silk suture was tightened
around the intraluminal filament to prevent bleeding. The
wound was then sutured and anesthesia discontinued. Sham
rats were exposed to the same surgical procedure without
occlusion of the MCA.
One hour after surgery, the animals were grossly assessed
for neurologic deficit as follows: 0 ¼ no deficit, 1 ¼ failure
to extend left forelimb, 2 ¼ decreased resistance to lateral
push, 3 ¼ circling to contralateral side, 4 ¼ walks only when
stimulated, and 5 ¼ no spontaneous motor activity. Only
rats with clear neurologic deficits (‡ 3), indicating successful
occlusion of the MCA [59], were included in the study.
To allow reperfusion, rats were briefly reanesthetized
with isoflurane, and the nylon filament was withdrawn 2 h
after MCAo. After the discontinuation of isoflurane and
wound closure, the animals were allowed to wake and were
kept in their cages with free access to food and water.
Neuropathology and quantification of ischemic
damage
Cerebral infarct volume was evaluated 22 h after reperfu-
sion in rats subjected to 2 h of MCAo. Rats were killed by

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

were then extracted [61], the organic phase was dried
under nitrogen, and the dry pellet was derivatized as
previously reported [62]. Briefly, 25 lLof10mm 4-(N-
chloroformylmethyl-N-methyl)amino-7-N,N-dimethyl-amino-
sulfonyl-2,1,3-benzoxadiazole (Tokyo Kasei Kogyo Co.,
Ltd, Tokyo, Japan) was added to 500 lL anhydrous
dichloromethane. The mixture was then heated at 60 °C
for 1 h, dried in a centrifugal concentrator (Martin Christ
GmbH, Osterode am Hartz, Germany), and reconstituted
in 50 lL of acetonitrile. HPLC with fluorimetric detection
was carried out using an S-200 fluorescence detector
(Perkin-Elmer Life Sciences). The separation was per-
formed with a mobile phase of acetonitrile ⁄ water (70 : 30,
v ⁄ v) at a flow rate of 1.0 mLÆmin
)1
. The concentration
of AEA was quantified by comparison with known
amounts of standard, as previously reported [61].
The hydrolysis of [
3
H]AEA by FAAH (EC 3.5.1.4)
was measured in rat brain areas (20 lg per test) by RP-
HPLC, using 10 lm [
3
H]AEA, as previously reported [63].
FAAH activity was expressed as pmol arachidonate
releasedÆmin
)1
Æ(mg protein)
)1

. 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

H]RTX was evaluated by rapid filtration
assays, performed as previously reported [66], and was
expressed as fmol RTX boundÆ(mg protein)
)1
. For both
agonists, the binding specificity was checked in the presence
of 1 lm ‘cold’ ligand [63,66].
The protein content of CB1 receptors and of FAAH was
quantified by ELISA, performed on brain homogenates
(20 lg per well) with polyclonal antibodies to CB1 receptor
(diluted 1 : 250) or FAAH (1 : 500) [63]. Goat anti-(rabbit
alkaline phosphatase) conjugate (diluted 1 : 2000) was used
as second antibody, and nonimmune rabbit serum (Primm
S.r.l) was used as a control for specificity.
Statistical analysis
Data are reported as means ± SD or means ± SEM, as
indicated. Statistical analysis was performed by the non-
parametric Mann–Whitney U -test, or by the unpaired Stu-
dent’s t-test (between two groups) or anova (for more than
two experimental groups), as indicated. Experimental data
were elaborated by means of the instat 3 program or the
prism 3 program (GraphPAD Software for Science, San
Diego, CA), and differences were considered statistically
significant when P < 0.05.
Acknowledgements
We wish to thank Drs Valeria Gasperi, Chiara De
Simone (University of Rome ‘Tor Vergata’), Natalia
Battista and Nicoletta Pasquariello (University of
Teramo) for their expert assistance with biochemical
analysis. Partial financial support from Ministero

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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