MINIREVIEW Post-ischemic brain damage: the endocannabinoid system in the mechanisms of neuronal death Domenico E Pellegrini-Giampietro1, Guido Mannaioni1 and Giacinto Bagetta2 Department of Preclinical and Clinical Pharmacology, University of Florence, Italy 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 FEBS Journal 276 (2009) 2–12 ª 2008 The Authors Journal compilation ª 2008 FEBS 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 (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 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 FEBS Journal 276 (2009) 2–12 ª 2008 The Authors Journal compilation ª 2008 FEBS The endocannabinoid system in cerebral ischemia D E Pellegrini-Giampietro et al Table 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 (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 after MCAO The protective effect of WIN 55212-2 was observed at 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 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 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 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 FEBS Journal 276 (2009) 2–12 ª 2008 The Authors Journal compilation ª 2008 FEBS 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 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 mgỈkg)1 rimonabant, but not with 0.3 or mgỈkg)1 rimonabant, produced a 50% reduction in infarct volume and a 40% improvement in neurological function in rats subjected to MCAO for h The protective effect was not observed with the CB agonist WIN 55212-2 (up to mgỈkg)1) and was associated with an increase in the brain content of anandamide A similar neuroprotection with 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 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 FEBS Journal 276 (2009) 2–12 ª 2008 The Authors Journal compilation ª 2008 FEBS The endocannabinoid system in cerebral ischemia D E Pellegrini-Giampietro et al beneficial effects in ischemic models so far is the compound AM251, which was able, at 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 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 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- FEBS Journal 276 (2009) 2–12 ª 2008 The Authors Journal compilation ª 2008 FEBS D E Pellegrini-Giampietro et al 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 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 (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 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 FEBS Journal 276 (2009) 2–12 ª 2008 The Authors Journal compilation ª 2008 FEBS The endocannabinoid system in cerebral ischemia D E Pellegrini-Giampietro et al 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 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 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Palmitoylethanolamide increases after focal cerebral ischemia and potentiates microglial cell motility J Neurosci 23, 7767–7775 FEBS Journal 276 (2009) 2–12 ª 2008 The Authors Journal compilation ª 2008 FEBS ... 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... 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. .. 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