Báo cáo khoa học: Post-ischemic brain damage: the endocannabinoid system in the mechanisms of neuronal death ppt

MINIREVIEW

Post-ischemic brain damage: the endocannabinoid system
in the mechanisms of neuronal death
Domenico E. Pellegrini-Giampietro1, Guido Mannaioni1 and Giacinto Bagetta2
1 Department of Preclinical and Clinical Pharmacology, University of Florence, Italy
2 Department of Pharmacobiology and University Center for Adaptive Disorders and Headache (UCADH), University of Calabria, Arcavacata
di Rende (CS), Italy

Keywords
ananadamide; 2-arachidonoylglycerol;
cannabinoids; CB receptors; cerebral
ischemia; endocannabinoids;
neuroprotection; neurotoxicity;
oxygen-glucose deprivation; stroke
Correspondence
D. E. Pellegrini-Giampietro, Department of
Pharmacology, University of Florence, Viale
Pieraccini 6, 50139 Firenze, Italy
Fax: +30 055 4271 280
Tel: +39 055 4271 205
E-mail: domenico.pellegrini@unifi.it
(Received 27 June 2008, revised 30
September 2008, accepted 24 October
2008)
doi:10.1111/j.1742-4658.2008.06765.x

An emerging body of evidence supports a key role for the endocannabinoid
system in numerous physiological and pathological mechanisms of the central nervous system. In the recent past, many experimental studies have
examined the putative protective or toxic effects of drugs interacting with
cannabinoid receptors or have measured the brain levels of endocannabinoids in in vitro and in vivo models of cerebral ischemia. The results of

these studies have been rather conflicting in supporting either a beneficial
or a detrimental role for the endocannabinoid system in post-ischemic neuronal death, in that cannabinoid receptor agonists and antagonists have
both been demonstrated to produce either protective or toxic responses in
ischemia, depending on a number of factors. Among these, the dose of the
administered drug and the specific endocannabinoid that accumulates in
each particular model appear to be of particular importance. Other mechanisms that have been put forward to explain these discrepant results are
the effects of cannabinoid receptor activation on the modulation of excitatory and inhibitory transmission, the vasodilatory and hypothermic effects
of cannabinoids, and their activation of cytoprotective signaling pathways.
Alternative mechanisms that appear to be independent from cannabinoid
receptor activation have also been suggested. Endocannabinoids probably
participate in the mechanisms that are triggered by the initial ischemic
stimulus and lead to delayed neuronal death. However, further information
is needed before pharmacological modulation of the endocannabinoid system may prove useful for therapeutic intervention in stroke and related
ischemic syndromes.

A wealth of information has accumulated to date concerning the basic mechanisms underlying post-ischemic
neuronal death in the mammalian brain. In the course
of cerebral ischemia (i.e. stroke, trauma, cardiac
arrest), abnormal levels of the excitatory amino acid
glutamate build up in the brain, causing ‘axon-sparing’
excitotoxic neuronal death. The recognized trigger for
such a devastating event is the excessive stimulation of

glutamate receptors, particularly of the ionotropic [i.e.
N-methyl-d-aspartate (NMDA)] subtype, which leads
to the accumulation of toxic amounts of intracellular
free calcium and of nitrogen and oxygen radical species, and to oxidative stress, committing the neuron to
death via activation of different downstream death
pathways selected in relation to the strength of the
detrimental stimulus [1]. This mechanism represents

Abbreviations
2-AG, 2-arachidonoylglycerol; AEA, anandamide; CB, cannabinoid; CNS, central nervous system; DAG, diacylglycerol; FAAH, fatty acid
amide hydrolase; GABA, 4-aminobutyrate; MCAO, middle cerebral artery occlusion; NMDA, N-methyl-D-aspartate; NO, nitric oxide; OGD,
oxygen-glucose deprivation; TRPV1, transient receptor potential vanilloid 1; D9-THC, D9-tetrahydrocannabinol.

2

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D. E. Pellegrini-Giampietro et al.

the rationale around which an intense area of pharmacological research has developed during the last
30 years, but which has failed to translate into clinically effective medicines [2]. Indeed, a large number of
clinical trials with neuroprotective drugs have yielded
disappointing results, from the use of NMDA receptor
antagonists to the most recent stroke-acute ischemic
NXY treatment II (SAINT II) clinical trial, in which a
promising free radical spin-trap was tested without
success [3].
A probable explanation for the failure of these trials
might be the dual role often played by mediators, such
as free radical species, that at physiological concentrations may be beneficial but which at high concentrations are detrimental for neuronal constituents. In fact,
the large amounts of nitric oxide (NO) generated by
pathological expression of NO synthase isoforms are
certainly neurotoxic, whereas homeostatic levels of NO
produced by the endothelial isoform of this enzyme
are beneficial by, among other mechanisms, sustaining
blood flow in the periphery of the ischemic brain. On

the other hand, under normal circumstances, stimulation of NMDA receptors is fundamental for physiological synaptic communication and strengthening [4] and,
hence, long-term blockade by competitive or noncompetitive NMDA antagonists, as is necessary for stroke
treatment, may be irrational [5]. The same reasoning
can be applied to the many other classes of anti-excitotoxic drugs tested thus far in clinical trials and certainly may provide the basis for other failures in the
future [6]. Therefore, a better design of protective
drugs and ⁄ or protocols for stroke treatment is needed,
together with the discovery of new molecular targets
for the development of innovative and effective therapeutic agents.
During the last decade a great deal of interest has
been devoted to dissecting the role of the endocannabinoid system in physiology as well as in pathological
processes. The system incorporates the endocannabinoids, their synthetic and degradative enzymes, the
endocannabinoid transporters and the cannabinoid
(CB) receptors, which include CB1 and CB2 receptors
as well as non-CB1 ⁄ CB2 receptors [e.g. transient
receptor potential vanilloid 1 (TRPV1) channels and
possibly others] [7–9]. The molecular cloning of two
seven-transmembrane-domain, G-protein (Gi ⁄ o)-coupled receptors termed CB1 [10] and CB2 [11], in conjunction with the availability of selective drugs, have
aided the comprehension of the neurobiology of this
system. CB1 receptors, which mediate the psychotropic
effects of D9-tetrahydrocannabinol (D9-THC) and
other CBs, are highly expressed in the central nervous
system (CNS) [12] whereas CB2 receptors are almost

The endocannabinoid system in cerebral ischemia

exclusively expressed in the immune system [13,14].
The best characterized endogenous ligands for CB1
receptors are N-arachidonoylethanolamide (AEA,
anandamide) [15] and 2-arachidonoylglycerol (2-AG)
[16–18], which are biosynthesized from membranederived lipid precursors by, respectively, the enzymes

N-acylphosphatidylethanolamine-hydrolyzing phospholipase D and diacylglycerol (DAG) lipase [8]. Because
of their lipid solubility, AEA and 2-AG cannot be
stored in vesicles and therefore they are synthesized on
demand and travel, in a retrograde direction, across
the postsynaptic membrane to the presynaptic membrane, where they activate presynaptic CB1 receptors
resulting in the inhibition of transmitter release
[19],probably via modulation of Ca2+ or K+ channels
[20,21]. Endocannabinoid uptake by central neurons
has been shown to be rapid, saturable, selective and
temperature dependent, implying the presence of a
membrane transporter for their facilitated diffusion
[22], although a specific transporter protein has yet to
be cloned. Once taken up into cells, AEA is degraded
by fatty acid amide hydrolase (FAAH) [23] and 2-AG
is degraded by monoacylglycerol lipase [24], although
the latter can also be metabolized by FAAH and other
recently identified lipases such as the ab-hydrolases 6
and 12 [8]. The endocannabinoid system in general,
and CB1 receptor-mediated presynaptic inhibition in
conjunction with endocannabinoid transport and
enzyme metabolism in particular, have been identified
as useful targets for neuroprotective drugs and have
been extensively studied in experimental models of
cerebral ischemia.

Endocannabinoids and CB receptors
in experimental models of cerebral
ischemia
In the past 10 years, numerous studies have addressed
the role of the endocannabinoid system in stroke and

in the mechanisms of post-ischemic neuronal death
(Table 1). To this end, models of focal and global
ischemia in vivo, with or without reperfusion, as well as
models of oxygen glucose deprivation (OGD) in neuronal culture preparations in vitro, have been utilized (a)
to investigate the putative protective or toxic effects of
drugs that interact with CB receptors or that inhibit
endocannabinoid catabolism or uptake, (b) to measure
the brain levels of the endocannabinoids AEA and
2-AG and (c) to explore the changes in gene expression
of CB1 and CB2 receptors. Earlier reports had shown
that D9-THC may be toxic when administered chronically to animals [25] but can also exert neuroprotective
and antioxidant effects against excitotoxicity in cortical

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The endocannabinoid system in cerebral ischemia

D. E. Pellegrini-Giampietro et al.

Table 1. The endocannabinoid system in experimental models of cerebral ischemia. 2VO, two-vessel occlusion; 4VO, four-vessel occlusion;
AEA, anandamide; 2-AG, 2-arachidonoylglycerol; CB, cannabinoid; CB-R, CB receptor; eCB, endocannabinoid; n.t., not tested; pMCAO,
permanent middle cerebral artery occlusion; tMCAO, transient middle cerebral artery occlusion. ›, increased; fl, decreased; =, no change.
Model
Transient global ischemia
Rat 4VO (15 min)
Rat hypotension + 2VO (12 min)
Gerbil 2VO (10 min)

Rat 4VO (7 min)
Gerbil 2VO (10 min)
Gerbil 2VO (10 min)
Focal ischemia
Rat pMCAO (24 h)
Rat tMCAO
Mouse tMCAO (20 min)
Rat tMCAO (1 h)
Rat pMCAO (72 h)
Mouse tMCAO (20 min)
Mouse tMCAO (4 h)
Rat tMCAO (2 h)
Rat pMCAO (5 h)
Rat pMCAO (5 h)
Rat tMCAO (2 h)
Mouse pMCAO (24 h)
Rat pMCAO (72 h)
Mouse tMCAO (1 h)
Mouse tMCAO (1 h)
Oxygen-glucose deprivation in vitro
Rat cortical neurons (8 h)
Rat cortical neurons (8 h)
Mouse midbrain slices (7 min)
Rat cortiscostriatal slices (30 min)
Hippocampal slices (15 min)

eCB levels ⁄ CB-R expression

CB-R activation

Protection
Protection
Protection
Protection
Toxicity
Protection

[28]
[30]
[31]
[37]
[48]
[38]

Protection
NT
Protection
Protection
Protection
NT
Protection
Toxicity
Toxicity
Toxicity
Toxicity
Protection
NT
Protection (CB2)
Toxicity

›CB1 (cortex)
CB1-KO mice

›AEA = 2-AG
›AEA = 2-AG
›AEA
=[3H]CP 55 940 binding
›AEA (striatum) late fl AEA (cortex)
›2-AG = AEA
›CB2 (microglia)
›CB1 & CB2

Protection
Protection
Protection
Protection
Toxicity

›2-AG = AEA
›CB1 = CB2

neurons in vitro [26]. Experimental research in the field
of ischemia was mainly prompted by observations indicating that CBs could attenuate glutamate-induced
injury by inhibiting glutamate release via presynaptic
CB1 receptors coupled to G-proteins and N-type
voltage-gated calcium channels [20,27].
An endogenous neuroprotective response
The first CB to be tested in models of cerebral ischemia was the synthetic cannabimimetic compound WIN
55212-2 [28]. In this report, the CB receptor agonist
was neuroprotective in rats subjected to either fourvessel occlusion for 15 min (a model of transient

global ischemia) or to permanent middle cerebral
artery occlusion (MCAO). The drug was administered
intraperitoneally prior to the ischemic insult in both
models, but it was effective in the focal ischemic
paradigm also when given up to 30 min after MCAO.
The protective effect of WIN 55212-2 was observed at
4

Reference

[28]
[29]
[35]
[32]
[33]
[105]
[34]
[44]
[46]
[47]
[45]
[39]
[40]
[41]
[53]
[28]
[36]
[37]
[42]
[52]

doses of 0.1–1 mgỈkg)1, but not at a dose of 3 mgỈkg)1,
and the protective effect appeared to be mediated by
CB1 because it was prevented by co-administration of
the antagonist rimonabant (or SR141716A). In the
same study, WIN 55212-2 was also tested in cortical
neurons exposed to OGD for 8 h, but neuroprotection
in vitro lacked stereoselectivity, was insensitive to CB1
and CB2 receptor antagonists, and was not mimicked
by D9-THC, suggesting a non-CB receptor-mediated
mechanism of action. When the same group observed
an increase in CB1 receptor expression in the penumbral boundary zone, starting at 2 h and persisting for
at least 72 h after a transient MCAO episode [29], this
finding was interpreted as an endogenous neuroprotective response. Subsequent reports appeared to corroborate this view, by demonstrating that natural and
synthetic CBs could attenuate neuronal injury in models of global [30,31] and focal [32–34] ischemia in vivo,
although, at least in models of permanent MCAO,
CB1-induced hypothermia appeared to contribute to

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D. E. Pellegrini-Giampietro et al.

neuroprotection [32,33]. Consistent with these findings,
CB1 receptor-deficient mice exhibited increased susceptibility to NMDA neurotoxicity, as well as increased
mortality and a larger infarct size following permanent
focal ischemia [35].
Experimental studies in vitro confirmed that the
endocannabinoids AEA and 2-AG may attenuate
OGD injury in cortical cells, although via CB1-independent and CB2-independent mechanisms [36], and

that the CB receptor agonist WIN 55212-2, at low
(3–30 nm) concentrations, but not at higher concentrations (100–1000 nm), prevented excessive membrane depolarization and delayed the onset of
depolarization block in ventral tegmental area dopaminergic neurons exposed to OGD [37]. In the latter
study, the CB1 antagonist AM281 and the DAGlipase inhibitor, O-3640, exacerbated the detrimental
effects of OGD in vitro by releasing glutamate in
excess, indicating that the increase in 2-AG levels
that was observed by these authors following OGD
may protect dopaminergic neurons through a mechanism similar to depolarization-induced suppression of
excitation (see below). A similar noxious effect was
demonstrated with another CB1 antagonist, rimonabant (1 mgỈkg)1 intravenously), on the outcome of
transient forebrain ischemia in rats [37]. Neuroprotective effects were also obtained in vivo with the
endocannabinoid transporter inhibitor AM404 [38]
and with the FAAH inhibitor URB597 [39], thus
suggesting the contribution of anandamide to the
beneficial effects of CBs observed in these models.
A role for CB2 receptors?
Although CB2 receptors are not expressed in neurons
and were generally believed to be absent from the brain,
it has been shown that CB2-positive macrophages,
deriving from resident microglia and ⁄ or invading
monocytes, appear in rat brain 3 days after hypoxia ⁄
ischemia or permanent MCAO [40]. The CB2 agonists
O-3853 and O-1966 have been shown to reduce the
infarct size and to improve the neurological score in
mice 24 h after a transient episode of MCAO [41],
indicating that activation of CB2 may be important in
reducing inflammatory responses that may lead to secondary injury following cerebral ischemia. In another
study, both CB1 and CB2 receptor agonists were able
to prevent the cellular damage, the efflux of lactate
dehydrogenase, the release of glutamate and tumor

necrosis factor-a, and the expression of inducible NO
synthase caused by OGD in cortico-striatal slices, but
only CB1 receptors (not CB2 receptors) were significantly increased following the ischemia-like insult [42].

The endocannabinoid system in cerebral ischemia

The ‘dark side’ of CBs
An independent line of research supports a contrasting, neurotoxic role for CB receptor activation in
ischemia, a role that was referred to as the ‘dark side’
of endocannabinoids in a report describing the toxic
effects of intracerebroventricular administration of
anandamide [43]. In these studies, neuroprotective
effects on post-ischemic neuronal death were provided
by CB1 receptor antagonists, and in particular by
rimonabant. Muthian et al. [44] showed that pretreatment with 3 mgỈkg)1 rimonabant, but not with 0.3 or
1 mgỈkg)1 rimonabant, produced a 50% reduction in
infarct volume and a 40% improvement in neurological function in rats subjected to MCAO for 2 h. The
protective effect was not observed with the CB agonist
WIN 55212-2 (up to 1 mgỈkg)1) and was associated
with an increase in the brain content of anandamide.
A similar neuroprotection with 3 mgỈkg)1 rimonabant
but not with WIN 55212-2 was reported in the same
model by Amantea et al. [45], who were able to correlate the persistent post-ischemic increase in the levels
of striatal anandamide with an increased activity of
N-acylposphatidylethanolamine-hydrolyzing phospholipase D and reduced activity and expression of FAAH.
Both the accumulation of anandamide (and of other
N-acylethanolamines) and the protective effects of
rimonabant (at 1 mgỈkg)1) were also observed in a rat
permanent MCAO model [46]: the CB1 antagonist,
however, was unable to counteract the elevation in

anandamide levels or the ischemic release of glutamate.
A subsequent study by the same group showed that
rimonabant was able to prevent the ischemic downregulation of NMDA receptors in the penumbra [47],
confirming that the protective effects of this CB1
receptor antagonist are unlikely to be related to an
anti-excitotoxic mechanism. A contribution of TRPV1
channels to rimonabant-induced neuroprotection has
been proposed by the observation that the TRPV1
antagonist capsazepine completely prevents the attenuation of CA1 pyramidal cell loss induced by rimonabant in gerbils subjected to transient forebrain
ischemia [48]. In this study, the protective effects of
rimonabant exhibited a bell-shaped curve, as previously observed for WIN 55212-2 [28,37], and were
observed at relatively low doses (0.25–0.5 mgỈkg)1)
compared with the results of other studies. To confirm
the crucial role of TRPV1 channels in neurodegenerative disorders [49], it is worth noting that capsazepine
has also been reported to prevent the neuroprotective
effects of the agonist capsaicin in models of global
ischemia [50] and ouabain-induced toxicity in vivo
[51]. The only other CB1 antagonist that has shown

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D. E. Pellegrini-Giampietro et al.

beneficial effects in ischemic models so far is the compound AM251, which was able, at 1 lm, to improve
markedly the post-OGD recovery of synaptic transmission in acute hippocampal slices [52]. In a very recent

study, the beneficial effects of rimonabant in a model
of focal ischemia were mimicked and potentiated by
the CB2 agonist O-1966 [53], suggesting that the
modulation of the balance between CB1 and CB2
receptor activities may represent an intriguing novel
possibility for ischemic therapeutic approaches.

2-AG, are known to activate and desensitize TRPV1
receptors (see below).
Numerous hypotheses have been put forward in the
past few years to reconcile these discrepant and controversial findings. In the following sections, we will
review some of the most important mechanisms that
have been proposed to date in an attempt to explain
the reasons whereby activation of CB receptors may
lead to either neuroprotection or neurotoxicity in
models of neurodegeneration and ischemia.

The endocannabinoid system in
cerebral ischemia – a neuroprotective
or a neurotoxic mechanism?

Modulation of excitatory and inhibitory
neurotransmission

The almost ubiquitous presence of the endocannabinoid machinery in every cell of the CNS, together with
the high level of CB1 receptor expression in critical
brain regions (cerebellum, hippocampus, neocortex
and basal ganglia), highlights the endocannabinoid system as an important modulator and possible pharmacological target for many physiological mechanisms
(i.e. learning, memory, appetite control, the reward
system) and pathological conditions, such as pain,

anxiety, mood disorders, motor disturbances and neurodegenerative diseases, including cerebral ischemia
[8,54,55]. As discussed, the scientific literature on neurodegenerative disorders, and specifically on ischemia
research (Table 1), has not always been consistent in
sustaining either a beneficial or a detrimental role for
the endocannabinoid system in the CNS [9,55–57]. CB
receptor agonists and antagonists have both been demonstrated to produce either protective or toxic
responses in ischemia, depending on a number of factors. Among these, two of the most important appear
to be (a) the dose of the administered CB drug and (b)
the specific endocannabinoid that accumulates in each
particular model. Indeed, in some studies, the CB agonist WIN 55212-2 appears to exert protective effects
in vivo at 0.1–1 mgỈkg)1 intraperitoneally but not at
higher doses [28,37], whereas the antagonist rimonabant displays neuroprotection at 0.25–0.5 mgỈkg)1 but
a certain degree of toxicity at 3 mgỈkg)1 [48]. Another
very striking feature emerging from the experimental
studies in models of cerebral ischemia is the fact that
when CB receptors mediate neurotoxicity (i.e. CB
receptor agonists are toxic and ⁄ or antagonists are protective) the endocannabinoid that is increased following ischemia is always AEA, and not 2-AG [44–46],
whereas the opposite appears to occur when CB receptors mediate neuroprotection [37,39] (Table 1). This
peculiar phenomenon may be a result of the fact that
AEA and other N-acethylethanolamines, but not
6

In neurons, CB1 receptors are mainly localized on
axon presynaptic terminals and thereby they play an
important role in the regulation of neurotransmitter
release [19,58]. More specifically, CB1 receptor activation by endocannabinoids has been shown to inhibit
either glutamatergic [59–62] or GABAergic [63,64] synaptic transmission, depending on the brain region,
through a presynaptic mechanism. The current ‘molecular logic’ on the endocannabinoid system signaling [7]
predicts that AEA and 2-AG are synthesized on
demand in the membrane of postsynaptic neurons,

then immediately released into the synaptic cleft where
they retrogradely diffuse to activate CB1 receptors on
presynaptic terminals, which eventually leads to inhibition of N-type calcium currents and suppression of cell
excitability and neurotransmitter release [65–67]
(Fig. 1). Indeed, this view is corroborated, at least for
2-AG, by the findings that DAG lipases are expressed
in the dendritic postsynaptic compartment [68],
whereas monoacylglycerol lipase is primarily a presynaptic enzyme [69]. Presynaptic CB1 receptor activation
in different brain areas has been associated with the
modulation of important synaptic plasticity phenomena, such as depolarization-induced suppression of
inhibition [66,70], depolarization-induced suppression
of excitation [67,71], persistent suppression of evoked
inhibitory postsynaptic currents [72] and inhibitory
long-term depression [73]. All of these CB1-mediated
mechanisms, often driven by a functional interaction
with metabotropic glutamate receptors, tightly regulate
the synaptic concentrations of either glutamate or
GABA, depending on the brain area. Hence, the differential inhibition of glutamate or GABA in various
experimental models of cerebral ischemia may be one
of the principal reasons whereby activation of CB
receptors may lead to either neuroprotection or neurotoxicity (Fig. 1). Interestingly, a similar mechanism has
been observed in different models of hippocampal
epileptic seizures: when endocannabinoids target gluta-

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The endocannabinoid system in cerebral ischemia

Glutamatergic
terminal

GABAergic
terminal

CB1

CB1
GA
B

A

Glutamate

Soma

AEA

2-AG
Spine

NAPE-PLD

DAG-L

Neurotoxicity

Neuroprotection

Fig. 1. Schematic model providing a hypothetic mechanism that
involves the modulation of GABAegic and glutamate release for the
dual toxic ⁄ protective role played by the endocannabinoid system in
post-ischemic neuronal death. At the postsynaptic membrane level,
the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol
(2-AG) are biosynthesized, respectively, by the enzymes N-acylphosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD)
and diacylglycerol lipase (DAG-L). Immediately after the synthesis
AEA and 2-AG are released into the synaptic cleft, from which they
diffuse retrogradely to activate presynaptic cannabinoid 1 (CB1)
receptors. Depending on the brain region or the experimental
model, CB1 receptors can be localized on the presynaptic terminals
of either GABAergic or glutamatergic neurons, promoting, alternatively, the suppression of the release of GABA, which is a potentially neurotoxic mechanism, or of glutamate, which instead may
lead to neuroprotection.

matergic neurons they provide neuroprotection [74,75],
whereas when they suppress GABAergic transmission
they enhance hyperexcitability [76,77].
Recently, a novel endocannabinoid–glutamate signaling pathway that may be of relevance in mediating
the physiological and pathological effects of CBs in
the hippocampus has been described [78]. This mechanism involves a neuron–astrocyte communication, in
which endocannabinoids released by neurons activate
CB1 receptors located in astrocytes, leading to phospholipase C-dependent Ca2+ mobilization from astrocytic internal stores, astrocytic release of glutamate
and eventually activation of NMDA receptors in pyramidal cells.

autoregulation and hence to an unfavorable outcome,
at least in MCAO models [82,83]. AEA and 2-AG may
also produce vasodilation through a TRPV1-mediated
mechanism [84], possibly involving the production of

NO from endothelial cells [85–87]. It should be noted,
however, that 2-AG was unable to reproduce the vasodilator response of AEA via TRPV1 receptors in
another study [88].
The reduction in brain temperature by both
D9-THC and synthetic CBs has been proposed as an
important possible mechanism underlying the neuroprotective effects of endocannabinoids. Warming the
animals to the body temperature of controls prevented
the neuroprotective effects of CB1 agonists in some
studies using models of focal [33,34] and global [38]
cerebral ischemia. However, it should be taken into
account that D9-THC was shown to be neuroprotective also at doses that were not hypothermic [38] or in
animals where temperature was under rigorous control
[30]. CB1 receptors located in the pre-optic anterior
hypothalamic nucleus have been suggested to be the
primary mediators of CB-induced hypothermia [89].
Activation of cytoprotective/anti-apoptotic
signaling pathways
Biochemical pathways that trigger apoptotic cell death
or cytoprotective cellular mechanisms can be differentially affected by CB receptor activation. Initially,
D9-THC was demonstrated to induce apoptosis in
cultured hippocampal neurons and slices [90]. More
recently, D9-THC and other CBs have revealed that
CB1 receptors are coupled, in a rimonabant-dependent
manner, to the anti-apoptotic phosphatidylinositol
3-kinase ⁄ Akt signaling pathway [91,92]. Activation of
this pathway appears to mediate the neuroprotective
effects of CBs in oligodendrocytes [93] and neurons
[94]. Furthermore, genetic suppression or pharmacological antagonism of CB1 receptors blocks the production of brain-derived neurotrophic factor following
toxic administration of kainic acid [74,95], suggesting
that brain-derived neurotrophic factor may be another

important mediator of the neuroprotective effects of
CBs.
CB receptor-independent mechanisms

Vasodilation and hypothermia
Activation of CB1 receptors in cerebral blood vessels
results in decreased vascular resistance and increased
blood flow [79–81]. CB receptor-mediated cerebral
vasodilation may have beneficial effects in ischemic
brain but may also lead to a loss of cerebrovascular

A number of potentially neuroprotective as well as
neurotoxic effects of CBs do not appear to be mediated by direct activation of CB receptors. For example,
some CBs, including D9-THC, possess antioxidant
properties and protect various cell types against oxidative stress [26,96], an effect that has been demonstrated

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to depend on the phenolic structure of the compounds
and not on their interaction with CB1 receptors [97].
Moreover, AEA and other N-acylethanolamines that
are known to accumulate in rodent models of permanent MCAO [39,46] may elicit biological cytotoxic
effects through targets other than CB receptors [43].

Among them, in mouse epidermal JB6 cells, AEA and
N-acylethanolamines stimulate CB-independent extracellular regulated kinase phosphorylation and, at
higher concentrations, have profound cytotoxic effects
owing to a collapse of mitochondrial energy metabolism, which compromises mitochondrial function [98].
One of the most important CB receptor-independent
mechanisms underlying the neurotoxic effects of CBs
might involve the activation of vanilloid receptors such
as TRPV1. AEA has been demonstrated to activate
TRPV1 channels both in vitro and in vivo and to upregulate genes involved in pro-inflammatory ⁄ microglialrelated responses [43,99,100]. In addition, AEA can
induce an acute release of NO through endothelial
TRPV1 activation [87], which may be responsible for
CB-induced vasorelaxation and hence has beneficial, but
also detrimental, effects (see above) in models of ischemia. It has been suggested that rimonabant, by blocking
CB1 receptors, leads to neuroprotection against excitotoxicity and ischemia because the increased concentrations of N-acylethanolamines, including AEA, activate
and desensitize TRPV1 receptors [48,51].
Recently, the G-protein-coupled receptor GPR55
has been proposed as a new CB receptor with signaling
pathways distinct from those of classical CB1 ⁄ CB2
receptors [101]. Activation of GPR55 increases
intracellular Ca2+ concentrations and inhibits M-type
K+-channel currents, thereby enhancing neuronal
excitability [101] and potentially toxic events if
expressed in neurons.

Concluding remarks
The great deal of knowledge accumulated in the past
three decades on the mechanisms underlying damage
inflicted to the brain tissue by cerebral ischemia has
failed to translate into effective medicines. Most
recently, a renewed interest towards molecular targets

for the development of novel stroke therapies has been
stimulated by the detailed description of the endocannabinoid system in the mammalian brain. This has been
accomplished thanks to the current availability of
drugs to target not only CB1 and CB2 receptors, but
also the biosynthesis, metabolism and transport of
endocannabinoids. As discussed, conflicting results
have accumulated with the use of drugs targeting CB1
receptors in models of cerebral ischemia, which may
8

depend on the experimental model, the dose of drug
administered and the specific endocannabinoid that
accumulates. Recent reviews have attempted to explain
these discrepancies by proposing that endocannabinoids may act as protective agents only in a time- and
space-specific manner, whereas they might contribute
to neurodegeneration if their action loses specificity
[8,102–104]. Probably, a more definitive role for CB2
receptor antagonists as anti-inflammatory drugs can be
anticipated, although the efficacy in the clinic settings
still awaits a conclusive demonstration. It is conceivable that in the course of cerebral ischemia, as documented in the recent past for other endogenous
targets, endocannabinoids participate in a complex
series of events initiated by the detrimental stimulus.
However, further information is needed before
pharmacological modulation of the endocannabinoid
system may prove useful for therapeutic intervention.

Acknowledgements
This work was supported by grants from the Italian
Ministry of University and Research (MIUR, PRIN
2006 project) to DEPG and GB, by the University of

Florence to DEPG and GM, and by the University of
Calabria to GB.

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