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
Trang 1Post-ischemic brain damage: the endocannabinoid system
in the mechanisms of neuronal death
Domenico E Pellegrini-Giampietro1, Guido Mannaioni1and 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
A wealth of information has accumulated to date
con-cerning 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 spe-cies, 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
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 cen-tral 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 endocannabi-noids 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 neu-ronal 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 mecha-nisms that have been put forward to explain these discrepant results are the effects of cannabinoid receptor activation on the modulation of excit-atory and inhibitory transmission, the vasodilexcit-atory 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 sys-tem may prove useful for therapeutic intervention in stroke and related ischemic syndromes
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.
Trang 2the rationale around which an intense area of
pharma-cological research has developed during the last
30 years, but which has failed to translate into
clini-cally 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
tions may be beneficial but which at high
concentra-tions 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,
stimula-tion of NMDA receptors is fundamental for
physiolog-ical synaptic communication and strengthening [4] and,
hence, long-term blockade by competitive or
noncom-petitive 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-excito-toxic drugs tested thus far in clinical trials and
cer-tainly 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
thera-peutic agents
During the last decade a great deal of interest has
been devoted to dissecting the role of the
endocannabi-noid system in physiology as well as in pathological
processes The system incorporates the
endocannabi-noids, 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)-cou-pled receptors termed CB1 [10] and CB2 [11], in
con-junction 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
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 membrane-derived lipid precursors by, respectively, the enzymes N-acylphosphatidylethanolamine-hydrolyzing phospho-lipase D and diacylglycerol (DAG) phospho-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 mem-brane, 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 neuro-nal 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 chroni-cally to animals [25] but can also exert neuroprotective and antioxidant effects against excitotoxicity in cortical
Trang 3neurons in vitro [26] Experimental research in the field
of ischemia was mainly prompted by observations
indi-cating 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
ische-mia was the synthetic cannabimimetic compound WIN
55212-2 [28] In this report, the CB receptor agonist
was neuroprotective in rats subjected to either
four-vessel 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
doses of 0.1–1 mgÆkg)1, but not at a dose of 3 mgÆkg)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 penum-bral 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 neuroprotec-tive response Subsequent reports appeared to corrobo-rate this view, by demonstrating that natural and synthetic CBs could attenuate neuronal injury in mod-els 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
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.
Transient global ischemia
Focal ischemia
Oxygen-glucose deprivation in vitro
Trang 4neuroprotection [32,33] Consistent with these findings,
CB1 receptor-deficient mice exhibited increased
suscep-tibility 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-inde-pendent and CB2-indeCB1-inde-pendent mechanisms [36], and
that the CB receptor agonist WIN 55212-2, at low
(3–30 nm) concentrations, but not at higher
concen-trations (100–1000 nm), prevented excessive
mem-brane depolarization and delayed the onset of
depolarization block in ventral tegmental area
dopa-minergic neurons exposed to OGD [37] In the latter
study, the CB1 antagonist AM281 and the
DAG-lipase 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
mecha-nism similar to depolarization-induced suppression of
excitation (see below) A similar noxious effect was
demonstrated with another CB1 antagonist,
rimona-bant (1 mgÆkg)1 intravenously), on the outcome of
transient forebrain ischemia in rats [37]
Neuropro-tective 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
sec-ondary 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
signifi-cantly increased following the ischemia-like insult [42]
The ‘dark side’ of CBs
An independent line of research supports a contrast-ing, 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 pretreat-ment with 3 mgÆkg)1 rimonabant, but not with 0.3 or
1 mgÆkg)1 rimonabant, produced a 50% reduction in infarct volume and a 40% improvement in neurologi-cal 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 mgÆkg)1) and was associated with an increase in the brain content of anandamide
A similar neuroprotection with 3 mgÆkg)1 rimonabant but not with WIN 55212-2 was reported in the same model by Amantea et al [45], who were able to corre-late the persistent post-ischemic increase in the levels
of striatal anandamide with an increased activity of N-acylposphatidylethanolamine-hydrolyzing phospholi-pase 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 mgÆkg)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 down-regulation 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 attenu-ation of CA1 pyramidal cell loss induced by rimona-bant in gerbils subjected to transient forebrain ischemia [48] In this study, the protective effects of rimonabant exhibited a bell-shaped curve, as previ-ously observed for WIN 55212-2 [28,37], and were observed at relatively low doses (0.25–0.5 mgÆkg)1) compared with the results of other studies To confirm the crucial role of TRPV1 channels in neurodegenera-tive 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
Trang 5beneficial effects in ischemic models so far is the
com-pound AM251, which was able, at 1 lm, to improve
markedly the post-OGD recovery of synaptic
transmis-sion 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
The endocannabinoid system in
cerebral ischemia – a neuroprotective
or a neurotoxic mechanism?
The almost ubiquitous presence of the
endocannabi-noid 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
sys-tem as an important modulator and possible
pharma-cological 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
neuro-degenerative diseases, including cerebral ischemia
[8,54,55] As discussed, the scientific literature on
neu-rodegenerative 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
dem-onstrated to produce either protective or toxic
responses in ischemia, depending on a number of
fac-tors 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
ago-nist WIN 55212-2 appears to exert protective effects
in vivo at 0.1–1 mgÆkg)1 intraperitoneally but not at
higher doses [28,37], whereas the antagonist
rimona-bant displays neuroprotection at 0.25–0.5 mgÆkg)1 but
a certain degree of toxicity at 3 mgÆkg)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
pro-tective) the endocannabinoid that is increased
follow-ing ischemia is always AEA, and not 2-AG [44–46],
whereas the opposite appears to occur when CB
recep-tors mediate neuroprotection [37,39] (Table 1) This
peculiar phenomenon may be a result of the fact that
AEA and other N-acethylethanolamines, but not
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 con-troversial 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
Modulation of excitatory and inhibitory neurotransmission
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 activa-tion by endocannabinoids has been shown to inhibit either glutamatergic [59–62] or GABAergic [63,64] syn-aptic transmission, depending on the brain region, through a presynaptic mechanism The current ‘molec-ular 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 inhibi-tion 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 presyn-aptic enzyme [69] Presynpresyn-aptic CB1 receptor activation
in different brain areas has been associated with the modulation of important synaptic plasticity pheno-mena, 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 dif-ferential 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 neuro-toxicity (Fig 1) Interestingly, a similar mechanism has been observed in different models of hippocampal epileptic seizures: when endocannabinoids target
Trang 6gluta-matergic neurons they provide neuroprotection [74,75],
whereas when they suppress GABAergic transmission
they enhance hyperexcitability [76,77]
Recently, a novel endocannabinoid–glutamate
sig-naling pathway that may be of relevance in mediating
the physiological and pathological effects of CBs in
the hippocampus has been described [78] This
mecha-nism involves a neuron–astrocyte communication, in
which endocannabinoids released by neurons activate
CB1 receptors located in astrocytes, leading to
phos-pholipase C-dependent Ca2+ mobilization from
astro-cytic internal stores, astroastro-cytic release of glutamate
and eventually activation of NMDA receptors in
pyra-midal cells
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
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 vaso-dilator 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 neuro-protective 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 neuroprotec-tive 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 differen-tially affected by CB receptor activation Inidifferen-tially, 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 pharmaco-logical antagonism of CB1 receptors blocks the pro-duction 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
A number of potentially neuroprotective as well as neurotoxic effects of CBs do not appear to be medi-ated by direct activation of CB receptors For example, some CBs, including D9-THC, possess antioxidant properties and protect various cell types against oxida-tive stress [26,96], an effect that has been demonstrated
Glutamatergic terminal
G A B A
Glutamate
CB 1
GABAergic
terminal
Soma
Spine
DAG-L NAPE-PLD
2-AG
AEA
CB 1
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-acyl-phosphatidylethanolamine-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,
alterna-tively, the suppression of the release of GABA, which is a
poten-tially neurotoxic mechanism, or of glutamate, which instead may
lead to neuroprotection.
Trang 7to 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
perma-nent 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
extra-cellular regulated kinase phosphorylation and, at
higher concentrations, have profound cytotoxic effects
owing to a collapse of mitochondrial energy
metabo-lism, 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
upre-gulate genes involved in pro-inflammatory⁄
microglial-related 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
ische-mia It has been suggested that rimonabant, by blocking
CB1 receptors, leads to neuroprotection against
excito-toxicity and ischemia because the increased
concentra-tions 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
endocann-abinoid 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
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 endocannabi-noids 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 conceiv-able that in the course of cerebral ischemia, as docu-mented 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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