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
Trang 1ischemia in the rat is involved in neuroprotection afforded
by 17b-estradiol
Diana Amantea1, Paola Spagnuolo1,2, Monica Bari2,3, Filomena Fezza2,3, Cinzia Mazzei1,
Cristina Tassorelli4, Luigi A Morrone1, Maria T Corasaniti3,5, Mauro Maccarrone3,6,* and
Giacinto Bagetta1,*
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: g.bagetta@unical.it
*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; E2, 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-arachidonoylphosphatidylN-acylphosphatidyl-ethanolamine; RTX, resinferatoxin; TRPV1, transient receptor potential vanilloid-1; TTC, 2,3,5-triphenyltetrazolium chloride.
Trang 2Endocannabinoids 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 Ca2+ 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 (E2) 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 E2 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 E2administration significantly reduces brain damage following transient MCAo [25]
Although the neuroprotective effects of E2 in humans are controversial [26], there is evidence that E2 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
E2 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 E2 via interaction with intracellular signaling cascades However, there is evidence documenting that neuroprotection may also occur via interaction with ER-like membrane 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 E2, 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
Trang 3modulation 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.
Trang 4The 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
E2 on endogenous AEA levels in both ischemic cortex and striatum was evaluated The results showed that
E2 (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 E21 h prior to MCAo (Fig 1B,C) It seems of further interest that, although brain ischemia did not alter cannabinoid receptor expression, E2 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, E2 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 E2 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
E2in the ischemic striatum seems to involve the activa-tion of intracellular ERs In fact, administraactiva-tion 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 E2(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.
0
50
100
150
200
Striatum Cortex
**
*
***
*,# *,##
0
Reperfusion (h)
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.
Trang 5ER antagonist ICI182 780 (0.25 mgÆkg)1,
intraperito-neal, 1 h prior to E2) was able to significantly
antago-nize the effects of E2 (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 E2 (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
E2 was reverted by the ER antagonist ICI182 780 (0.25 mgÆkg)1, intraperitoneal), administered 1 h prior
to E2(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 E2(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 E2 Endogenous AEA [pmolÆ(mg protein))1] 35 ± 3 30 ± 9
FAAH activity [pmolÆmin)1Æ(mg protein))1] 820 ± 80 884 ± 90
NAPE-PLD activity [pmolÆmin)1Æ(mg protein))1] 22 ± 3 25 ± 3
CB1 receptor binding [fmolÆ(mg protein))1] 190 ± 20 180 ± 20
Trang 6contribute 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 E2on 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 + E2;
& P < 0.05 versus MCAo + E2.
Vehicle E2 ICI + E2 0
200 400 600
**
0 1 2 3 4 5 6 7 8 0
25 50 75
B
E2 ICI + E2
coronal section
Fig 6 Neuroprotection afforded by E2against 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.
Trang 7suggested 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 modulainforma-tion of the endocannabi-noid system by E2 in the brain under pathophysiologic conditions Here, we show that acute administration of
a pharmacologic dose of E2 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 E2 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 E2 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
Trang 8Experimental procedures
Materials
Chemicals were of the purest analytical grade AEA,
resinferatoxin (RTX), E2 and R-(+)-WIN55,212-2 were
obtained from Sigma Chemical Co (St Louis, MO)
ICI182 780 was purchased from Tocris Bioscience
(Avonmouth, UK) [3H]AEA (223 CiÆmmol)1), [3H]RTX
(43 CiÆmmol)1) and [3H]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
22C, a relative humidity of 65%, and a 12 h light : 12 h
dark cycle, with free access to food and water E2was
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 E2 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 37C 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: 0¼ no deficit, 1 ¼ failure
to extend left forelimb, 2¼ decreased resistance to lateral push, 3¼ circling to contralateral side, 4 ¼ walks only when stimulated, and 5¼ 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 37C After 10 min of incubation, the slices were transferred to 10% neutral buffered formaldehyde and stored at 4C 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 (mm3) 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
Trang 9ipsilateral 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 lL of 10 mm
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 60C
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 [3H]AEA by FAAH (EC 3.5.1.4)
was measured in rat brain areas (20 lg per test) by
RP-HPLC, using 10 lm [3H]AEA, as previously reported [63]
FAAH activity was expressed as pmol arachidonate
releasedÆmin)1Æ(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 test1), using 100 lm N-[3
H]Arachi-donoyl-phosphatidylethanolamine, as previously reported
[64] NAPE-PLD activity was expressed as pmol AEA
releasedÆmin)1Æ(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 [3H]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 [3H]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 prism3 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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