Modulation of the endocannabinoid system by focal brain ischemia in the rat is involved in neuroprotection afforded by 17b-estradiol Diana Amantea 1 , Paola Spagnuolo 1,2 , Monica Bari 2,3 , Filomena Fezza 2,3 , Cinzia Mazzei 1 , Cristina Tassorelli 4 , Luigi A. Morrone 1 , Maria T. Corasaniti 3,5 , Mauro Maccarrone 3,6, * and Giacinto Bagetta 1, * 1 Department of Pharmacobiology and University Center for the Study of Adaptive Disorder and Headache (UCADH), Section of Neuropharmacology of Normal and Pathological Neuronal Plasticity, University of Calabria, Rende (CS), Italy 2 Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Rome, Italy 3 IRCCS Neurological Institute C. Mondino Foundation, Mondino-Tor Vergata Center for Experimental Neuropharmacology, Laboratory of Neurochemistry, Rome, Italy 4 Laboratory of Pathophysiology of Integrative Autonomic Systems, IRCCS Neurological Institute C. Mondino Foundation and University Centre for the Study of Adaptive Disorder and Headache (UCADH), Pavia, Italy 5 Department of Pharmacobiological Sciences, University Magna Graecia of Catanzaro, Italy 6 Department of Biomedical Sciences, University of Teramo, Italy Keywords endocannabinoids; estrogen; middle cerebral artery occlusion; stroke Correspondence G. Bagetta, Department of Pharmacobiology, University of Calabria, via P. Bucci Ed. Polifunzionale, 87036 Rende (CS), Italy Fax: +39 0984 493462 Tel: +39 0984 493462 E-mail: g.bagetta@unical.it *These authors contributed equally to this work (Received 28 March 2007, revised 18 June 2007, accepted 3 July 2007) doi:10.1111/j.1742-4658.2007.05975.x Endogenous levels of the endocannabinoid anandamide, and the activities of the synthesizing and hydrolyzing enzymes, i.e. N-acylphosphatidyletha- nolamine-hydrolyzing phospholipase D and fatty acid amide hydrolase, respectively, were determined in the cortex and the striatum of rats sub- jected to transient middle cerebral artery occlusion. Anandamide content was markedly increased ( 3-fold over controls; P < 0.01) in the ischemic striatum after 2 h of middle cerebral artery occlusion, but not in the cortex, and this elevation was paralleled by increased activity of N-acylphosphati- dylethanolamine-hydrolyzing phospholipase D ( 1.7-fold; P < 0.01), and reduced activity ( 0.6-fold; P < 0.01) and expression ( 0.7-fold; P < 0.05) of fatty acid amide hydrolase. These effects of middle cerebral artery occlusion were further potentiated by 1 h of reperfusion, whereas anandamide binding to type 1 cannabinoid and type 1 vanilloid receptors was not affected significantly by the ischemic insult. Additionally, the can- nabinoid type 1 receptor antagonist SR141716, but not the receptor agonist R-(+)-WIN55,212-2, significantly reduced (33%; P < 0.05) cerebral infarct volume detected 22 h after the beginning of reperfusion. A neuroprotective intraperitoneal dose of 17b-estradiol (0.20 mgÆkg )1 ) that reduced infarct size by 43% also minimized the effect of brain ischemia on the endocanna- binoid system, in an estrogen receptor-dependent manner. In conclusion, we show that the endocannabinoid system is implicated in the pathophysi- ology of transient middle cerebral artery occlusion-induced brain damage, and that neuroprotection afforded by estrogen is coincident with a re- establishment of anandamide levels in the ischemic striatum through a mechanism that needs to be investigated further. Abbreviations AEA, anandamide (arachidonoylethanolamide); CB, cannabinoid; CNS, central nervous system; E 2 ,17b-estradiol; ER, estrogen receptor; FAAH, fatty acid amide hydrolase; MCA, middle cerebral artery; MCAo, middle cerebral artery occlusion; NAPE, N-acylphosphatidyl- ethanolamine; NAPE-PLD,N-acylphosphatidylethanolamine-hydrolyzing phospholipase D; NArPE, N-arachidonoylphosphatidylethanolamine; RTX, resinferatoxin; TRPV1, transient receptor potential vanilloid-1; TTC, 2,3,5-triphenyltetrazolium chloride. 4464 FEBS Journal 274 (2007) 4464–4475 ª 2007 The Authors Journal compilation ª 2007 FEBS Endocannabinoids are amides, esters and ethers of long-chain polyunsaturated fatty acids that are synthe- sized on demand. Anandamide (arachidonoylethanol- amide) (AEA) was the first member of this family to be discovered [1], and it is synthesized by the enzyme N-acylphosphatidylethanolamine (NAPE)-hydrolyzing phospholipase D (NAPE-PLD) [2]. Following cellular depolarization and Ca 2+ influx, endocannabinoids are released into the extracellular space and interact with type 1 and type 2 cannabinoid (CB1 and CB2) recep- tors, non-CB1 ⁄ non-CB2 receptors, and noncannabi- noid receptors, including the type 1 vanilloid receptor [transient receptor potential vanilloid-1 (TRPV1)], a ligand-gated and nonselective cationic channel [3]. The biological actions of AEA cease following cellular uptake, mediated by a membrane transporter [4], and subsequent intracellular degradation catalyzed by a fatty acid amide hydrolase (FAAH), which cleaves the amide bond to form arachidonic acid and ethanol- amine [5]. Taken together, AEA, its congeners and the proteins that bind, synthesize or transport them form the ‘endocannabinoid system’ [6]. In the brain, endocannabinoids act as retrograde messengers to control multiple central nervous system (CNS) functions, including learning and memory, pain, sleep, and appetite [7]. Moreover, there is experimental evidence to support a dual role for AEA in the CNS as a neuroprotective or neurotoxic agent [8,9]. Endo- cannabinoids are indeed elevated in a variety of acute neurodegenerative insults, such as decapitation-induced ischemia [10], N-methyl-d-aspartate (NMDA)-induced excitotoxicity [11], convulsions [12], traumatic brain injury [13], and notably middle cerebral artery (MCA) occlusion (MCAo) [14,15]. This elevation has been sug- gested to represent an endogenous protective mecha- nism during CNS injury [16]. By contrast, recent studies have suggested that endogenously released en- docannabinoids may be toxic to neurons in animal models of acute neurodegeneration. Thus, for instance, both CB1 receptor stimulation and blockade have been shown to exert neuroprotection in rodent models of focal brain ischemia [14,15,17]. Recent studies have highlighted the ability of estro- gens to enhance recovery from ischemic brain injury resulting from cardiovascular disease or cerebrovascu- lar stroke. 17b-estradiol (E 2 ) has been shown to reduce mortality and cerebral damage in a variety of animal models of acute cerebral ischemia, including transient and permanent MCAo [18–20], photothrombotic focal ischemic brain damage [21], and global forebrain ische- mia [22,23]. Accordingly, administration of either pharmacologic or physiologic doses of E 2 provides neuroprotection in ovariectomized female rodents subjected to focal brain ischemia [18–20,24]. Similar results have been obtained in male rats, as either acute or chronic E 2 administration significantly reduces brain damage following transient MCAo [25]. Although the neuroprotective effects of E 2 in humans are controversial [26], there is evidence that E 2 enhances recovery from brain injury following cerebral ischemia [27,28], and continued use of estrogens has been shown to significantly reduce the risk of stroke [29–31]. This is also confirmed by epidemiologic evi- dence indicating that women are more protected than men against stroke until the menopause [27]. However, recent large, randomized, clinical trials have questioned the effectiveness of female sex hormones in the preven- tion of coronary heart disease and stroke [32–34]. Several mechanisms have been suggested to underlie E 2 neuroprotection, including modulation of synapto- genesis, protection against apoptosis, anti-inflamma- tory activity, and increased cerebral blood flow. Estrogens exert their activity through the interaction with intracellular estrogen receptors (ERs), ERa and ERb, which results in the modulation of the transcrip- tion of estrogen target genes, including those impli- cated in neuronal survival. ER activation may also mediate rapid nongenomic effects of E 2 via interaction with intracellular signaling cascades. However, there is evidence documenting that neuroprotection may also occur via interaction with ER-like membrane recep- tors, mediating rapid, nongenomic actions, or recep- tor-independent mechanisms, mainly due to the antioxidant free radical-scavenging properties of the steroidal molecules [35]. However, the exact contribu- tion of each molecular mechanism to the overall neu- rotrophic and neuroprotective effect of estrogens is still a matter of debate. Interestingly, recent studies have revealed that sex hormones may provide pivotal modulation of the endocannabinoid system in a tissue- and species-spe- cific manner, as demonstrated both in vivo, in mouse uterus, and in vitro, in human endothelial, lymphoma and neuroblastoma cells [36,37]. In particular, the endocannabinoid AEA is released from human endo- thelial cells treated with E 2 , and complements some actions of this hormone on human platelets [38]. However, the modulation of the endocannabinoid system by estrogen in the brain has been poorly investigated. In the present study, we aimed to evaluate the effect of MCAo-induced brain insult on AEA regional level, metabolism, and receptor binding and expression. The putative neuroprotective action of agonists and antagonists of cannabinoid receptors has also been investigated. Moreover, we demonstrate here that D. Amantea et al. Endocannabinoid system modulation by E 2 after MCAo FEBS Journal 274 (2007) 4464–4475 ª 2007 The Authors Journal compilation ª 2007 FEBS 4465 modulation of the endocannabinoid system is implicated in the mechanisms of neuroprotection afforded by acute administration of a pharmacologic dose of estrogen in male rats. Results Two hours of MCAo resulted in a significant increase in endogenous AEA levels in the striatum ipsilateral to the ischemic damage, but not in the cerebral cortex. Inter- estingly, when reperfusion was allowed for 1 h following 2 h of MCAo, endogenous levels of AEA were higher than those detected in the striata of rats subjected to brain ischemia without reperfusion (Fig. 1A). In order to evaluate whether ischemia-induced changes in endogenous AEA levels were associated with altered endocannabinoid metabolism, the activity of FAAH was measured in cortices and striata from rats with focal brain ischemia. Two hours of MCAo, with or without 1 h of reperfusion, resulted in a significant decrease in FAAH activity as detected in the striatum, but not in the cortex, ipsilateral to the ischemic damage (Fig. 1B). Furthermore, increased AEA levels in the ischemic striatum were also associated with a significant increase in NAPE-PLD activity, as detected following 2 h of MCAo (Fig. 1C). More interestingly, re-estab- lishment of the blood supply for 1 h resulted in a more pronounced increase in the activity of NAPE-PLD, as compared to the enzymatic activity measured in striata after 2 h of MCAo without reperfusion (Fig. 1C). By contrast, focal brain ischemia did not appear to affect NAPE-PLD activity in the cerebral cortex, and this is consistent with the lack of significant changes in endogenous AEA levels detected in this ischemic corti- cal region (Fig. 1A). The increase in endogenous AEA levels detected in the striatum was persistent also at later stages of reper- fusion following 2 h of MCAo (Fig. 2). By contrast, cortical levels of AEA, which did not significantly change after 1 h of reperfusion, were significantly reduced 6 h or 22 h later (Fig. 2). Unlike endocannabinoid metabolism, which appears to be modified as a consequence of focal brain ische- mia, CB1 and TRPV1 receptor binding in cortices and striata did not change following 2 h of MCAo, either in the absence or in the presence of 1 h of reperfusion (data not shown). Fig. 1. Endogenous levels of AEA (A) and activity of FAAH (B) and NAPE-PLD (C) in the ischemic striatum and cortex of rats subjected to 2 h of MCAo, with or without 1 h of reperfusion. Sham rats were exposed to the same surgical procedure without occlusion of the MCA. E 2 (0.20 mgÆkg )1 , intraperitoneal) was administered 1 h before MCAo. Values are expressed as mean ± SD (n ¼ 3), and were analyzed by the Mann–Whitney U-test. *P < 0.01 versus Sham; # P < 0.01 versus MCAo; § P < 0.05 versus MCAo. Endocannabinoid system modulation by E 2 after MCAo D. Amantea et al. 4466 FEBS Journal 274 (2007) 4464–4475 ª 2007 The Authors Journal compilation ª 2007 FEBS The lack of change in CB1 receptor binding capacity following MCAo was also confirmed by data showing that CB1 receptor striatal content was not modified by focal ischemic insult (Fig. 3). By contrast, striatal con- tent of the metabolic enzyme FAAH was significantly reduced following 2 h of MCAo, with or without 1 h of reperfusion (Fig. 3). The latter finding is consistent with the reduced activity of FAAH in the ischemic stri- atum of rats that have undergone MCAo (Fig. 1B). The lack of specific antibodies to NAPE-PLD pre- vented us from further extending the analysis of pro- tein content to this enzyme. In order to evaluate whether increased AEA levels following MCAo might contribute to ischemic brain damage or, conversely, might serve as an endogenous neuroprotective mechanism, we assessed the effect of CB1 receptor blockade or activation on ischemic dam- age. We found that administration of the CB1 recep- tor antagonist SR141716 (3 mgÆkg )1 , intraperitoneal), 15 min before MCAo, resulted in a significant reduc- tion in brain infarct volume as detected 22 h after rep- erfusion (Fig. 4A–C). By contrast, pretreatment with the cannabinoid receptor agonist R-(+)-WIN-55,212-2 (1 mgÆkg )1 , intraperitoneal, 15 min before MCAo) did not affect brain infarct damage produced by transient MCAo (Fig. 4D). Estrogens are known to protect the brain against focal ischemia [35]. In order to investigate the role of the endocannabinoid system in the neuroprotection afforded by estrogen, the effect of acute treatment with E 2 on endogenous AEA levels in both ischemic cortex and striatum was evaluated. The results showed that E 2 (0.20 mgÆkg )1 , intraperitoneal) administered 1 h before MCAo significantly reversed the increase of endogenous AEA levels produced by 2 h of focal cere- bral ischemia in the striatum (Fig. 1A). Moreover, FAAH and NAPE-PLD activities returned to basal (sham) levels when rats were treated with the same dose of E 2 1 h prior to MCAo (Fig. 1B,C). It seems of further interest that, although brain ischemia did not alter cannabinoid receptor expression, E 2 pretreatment resulted in a significant (45%) reduction of CB1 bind- ing in the striatum, but not in the cortex ipsilateral to the ischemic insult (data not shown). Instead, CB1 receptor content was not affected by the hormone treatment (Fig. 3), and neither was TRPV1 binding (data not shown). Interestingly, E 2 does not appear to significantly modulate basal levels of AEA, FAAH and NAPE- PLD activity and CB1 receptor binding as assessed in striatal samples from sham-operated rats, pretreated with E 2 or vehicle, 3 h before sacrifice (Table 1). This suggests that neuropathologic alterations of the endoc- annabinoid system, such those detected after MCAo, are instrumental for its modulation by estrogen. The modulation of the endocannabinoid system by E 2 in the ischemic striatum seems to involve the activa- tion of intracellular ERs. In fact, administration of the Fig. 3. FAAH and CB1 receptor content in the striatum of rats sub- jected to 2 h of MCAo, with or without 1 h of reperfusion. Sham rats were exposed to the same surgical procedure without occlusion of the MCA. E 2 (0.20 mgÆkg )1 , intraperitoneal) was administered 1 h before MCAo. Values are expressed as mean ± SD (n ¼ 4), and were analyzed by the Mann–Whitney U-test. **P < 0.05 versus Sham; § P < 0.05 versus MCAo. 1.0 6.0 22.0 0 50 100 150 200 Striatum Cortex ** * *** * ,# * ,## 0 Reperfusion (h) Endogenous levels of AEA (% of control) Fig. 2. Endogenous levels of AEA in the striatum and cortex of rats subjected to 2 h of MCAo, followed by 0, 1, 6 or 22 h of reperfu- sion (100% as MCAo samples in Fig. 1A). Values are expressed as mean ± SD (n ¼ 3), and were analyzed by the Mann–Whitney U-test. *P < 0.05, **P < 0.01 and ***P < 0.001 versus 0 h of reperfusion; # P < 0.01 and ## P < 0.001 versus 1 h of reperfusion. D. Amantea et al. Endocannabinoid system modulation by E 2 after MCAo FEBS Journal 274 (2007) 4464–4475 ª 2007 The Authors Journal compilation ª 2007 FEBS 4467 ER antagonist ICI182 780 (0.25 mgÆkg )1 , intraperito- neal, 1 h prior to E 2 ) was able to significantly antago- nize the effects of E 2 (0.20 mgÆkg )1 , intraperitoneal, 1 h before MCAo) on endogenous levels of AEA, on FAAH and NAPE-PLD activity, and on CB1 receptor binding in the striatum (Fig. 5). Interestingly, acute treatment with E 2 (0.20 mgÆkg )1 , intraperitoneal), given 1 h before the ischemic insult, resulted in a significant reduction of brain infarct volume produced by 2 h of MCAo followed by 22 h of reperfusion. The neuroprotection afforded by E 2 was reverted by the ER antagonist ICI182 780 (0.25 mgÆkg )1 , intraperitoneal), administered 1 h prior to E 2 (Fig. 6). Discussion The results reported in the present study demonstrate that a focal ischemic brain insult produced by transient MCAo results in a significant increase of endogenous AEA levels in the ischemic striatum, as early as 2 h following injury. This effect was associated with altered endocannabinoid metabolism, as 2 h of MCAo also resulted in reduced activity and expression of the metabolic enzyme FAAH, whereas NAPE-PLD activ- ity was significantly increased. Interestingly, we observed that reperfusion increased striatal AEA levels above those detected after 2 h of MCAo, thus suggest- ing that re-establishment of blood supply may further Fig. 4. SR141716, a selective CB1 receptor antagonist, but not WIN55,212-2, a CB1 receptor agonist, reduces brain infarct size following transient MCAo. The right MCA was occluded for 2 h with a nylon suture, as described in Experimental procedures, and cerebral infarct vol- ume was evaluated 22 h after reperfusion. Eight serial sections from each brain were cut at 2 mm intervals from the frontal pole and incu- bated in TTC, which stains viable tissue red but not infarcted areas (C). The infarct volume was calculated by summing the infarcted area of the eight sections (A) and multiplying by the interval thickness between sections. Rats received vehicle (vegetable oil, n ¼ 5) or SR141716 (3 mgÆkg )1 , n ¼ 4) intraperitoneally, 15 min prior to MCAo (A–C). In another set of experiments, rats received vehicle (propylene glycol, n ¼ 7) or WIN55,212-2 (1 mg kg )1 , n ¼ 7) intraperitoneally, 15 min prior to MCAo (D).Values are expressed as mean ± SEM, and were compared by unpaired two-tailed t-test. *P < 0.05 versus vehicle. Table 1. Effect of acute administration of E 2 on the endocannabi- noid system in striatal tissue from sham-operated rats. Rats were treated with E 2 (0.2 mgÆkg )1 , intraperitoneal) or vehicle (vegetable oil, 1 mLÆkg )1 , intraperitoneal), 3 h before sham operation. Values are expressed as mean ± SD (n ¼ 3), and were analyzed by the Mann–Whitney U-test. Vehicle E 2 Endogenous AEA [pmolÆ(mg protein) )1 ] 35±3 30±9 FAAH activity [pmolÆmin )1 Æ(mg protein) )1 ] 820 ± 80 884 ± 90 NAPE-PLD activity [pmolÆmin )1 Æ(mg protein) )1 ] 22±3 25±3 CB1 receptor binding [fmolÆ(mg protein) )1 ] 190 ± 20 180 ± 20 Endocannabinoid system modulation by E 2 after MCAo D. Amantea et al. 4468 FEBS Journal 274 (2007) 4464–4475 ª 2007 The Authors Journal compilation ª 2007 FEBS contribute to endocannabinoid modulation. The latter hypothesis is supported by the evidence that the increase in NAPE-PLD activity was more pronounced following 1 h of reperfusion, as compared to the enzy- matic activity measured after MCAo alone. Thus, it is conceivable that an early increase in endogenous AEA levels in the ischemic striatum, which comprises most of the ischemic core [39], might underlie brain damage produced by focal ischemia. This effect appears to occur via activation of cannabinoid receptors, as pre- treatment with the CB1 receptor antagonist SR141716 afforded neuroprotection in rats subjected to transient MCAo. An early increase of AEA has been previously reported in the whole brain of rats following transient focal brain ischemia [15]. However, in that study, no information was collected about the alterations induced by the ischemic insult in different brain regions, and neither was the biochemical background behind the effect of MCAo on AEA levels investigated [15]. We did observe an early significant increase in endogenous AEA levels in the ischemic striatum but not in the cortex of rats subjected to MCAo. The lack of acute changes in endocannabinoid levels in the cortical regions may stem from differential regional susceptibility to the ischemic insult, 2 h of MCAo being not enough to produce significant AEA elevation in the penumbral region. By contrast, we did observe a reduction in AEA levels in the cortex at later stages of reperfusion, which may indeed be the result of delayed damage, as compared to the striatum [39]. However, the exact pathophysiologic significance of the latter observation needs to be investigated further. Endogenous levels of AEA are elevated by decapita- tion-induced ischemia [10], NMDA-induced excitotox- icity in vivo [11], neonatal traumatic brain injury [11], kainate-induced neuronal excitation [40] and, most notably, MCAo [15]. This elevation of AEA has been Fig. 5. The observed effects of E 2 on endogenous levels of AEA, on FAAH and NAPE-PLD activity, and on CB1 receptor binding in the striatum of rats following MCAo appear to be mediated by E 2 receptor stimulation, as these effects are reversed by ICI182 780, a pure ER antagonist. Values are expressed as mean ± SD (n ¼ 4), and analyzed by the Mann–Whitney U-test. # P < 0.01 versus MCAo; § P < 0.05 versus MCAo; @ P < 0.01 versus MCAo + E 2 ; & P < 0.05 versus MCAo + E 2 . Vehicle E 2 ICI + E 2 0 200 400 600 ** Infarct volume (mm 3 ) 0 1 2 3 4 5 6 7 8 0 25 50 75 B A Vehicle E 2 ICI + E 2 coronal section Infarct area (mm 2 ) Fig. 6. Neuroprotection afforded by E 2 against brain damage pro- duced by transient MCAo is reversed by ICI182 780, a pure ER antagonist. The right MCA was occluded for 2 h with a nylon suture, as described in Experimental procedures, and cerebral infarct volume was evaluated 22 h after reperfusion. Eight serial sections from each brain were cut at 2 mm intervals from the fron- tal pole and incubated in TTC, which stains viable tissue red but not infarcted areas. The infarct volume (B) was calculated by sum- ming the infarcted area of the eight sections (A) and multiplying by the interval thickness between sections. Rats received E 2 (0.20 mgÆkg )1 , intraperitoneal, 1 h before MCAo), alone or in combi- nation with ICI182 780 (0.25 mgÆkg )1 , intraperitoneal, 1 h prior to E 2 ). Values are expressed as mean ± SEM (n ¼ 5), and were ana- lyzed by ANOVA followed by Tukey’s post hoc test. **P < 0.01 ver- sus vehicle. D. Amantea et al. Endocannabinoid system modulation by E 2 after MCAo FEBS Journal 274 (2007) 4464–4475 ª 2007 The Authors Journal compilation ª 2007 FEBS 4469 suggested to represent an endogenous protective mecha- nism during CNS injury [16]. In line with this, exo- genously administered (endo)cannabinoids have been shown to protect neurons via several mechanisms, yet the role of endogenously released endocannabinoids on neuronal damage appears to be controversial [9]. In fact, recent studies have paradoxically suggested that endogenously released endocannabinoids may be toxic to neurons in animal models of acute neurodegenera- tion. Accordingly, administration of the CB1 receptor antagonist SR141716 evoked a significant neuroprotec- tive response in adult rats subjected to permanent or transient MCAo [14,15], and in neonatal rats exposed to an intrastriatal microinjection of NMDA [41]. This is consistent with our data, documenting that systemic administration of SR141716 results in a significant reduction of brain infarct volume produced by transient MCAo, thus suggesting that increased AEA levels pro- duced during the early stages of brain ischemic insult may trigger neurodegenerative events through activa- tion of CB1 receptors. It seems noteworthy that, despite the acute neuronal injury that occurs in the ischemic striatum following MCAo, under the present experi- mental conditions CB1 receptor expression and ligand- binding capacity are not compromised. CB1 receptors are predominantly localized on presynaptic nerve termi- nals, and their stimulation can elicit either inhibitory effects by blocking glutamate release or excitatory effects by blocking 4-aminobutyric acid (GABA) release, depending on which neuronal circuits are acti- vated [7,42]. Although inhibition of glutamate release has been suggested to represent a pivotal mechanism involved in endocannabinoid-mediated neuroprotection [17,43–46], CB1 receptor-induced reduction of the inhibitory GABAergic input in the striatum [47] may conversely provide a mechanism underling neurodegen- eration. Moreover, activation of CB1 receptors local- ized on cerebral blood vessels has been suggested to determine altered autoregulation of cerebral blood flow [48–50], and this may further contribute to brain dam- age following the ischemic insult. Thus, although it can- not be excluded that AEA may be neurotoxic via activation of molecular targets distinct from CB1, our data suggest that neurotoxicity occurs through CB1 receptor activation. Accordingly, cannabinoid receptor activation may induce [51] or prevent [52] apoptosis, implying that CB1 receptors represent a key regulator of cell survival ⁄ death and a useful pharmacologic target to control cell death in neurodegenerative diseases. Increased levels of N-acylethanolamines following brain injury have been suggested to depend on accumulation of the corresponding precursors NAPE [11,14]. Here, we found that the activity of the AEA-synthesizing enzyme NAPE-PLD was signifi- cantly increased following MCAo, and that this was paralleled by a significant reduction in the activity and expression of the AEA-hydrolyzing enzyme FAAH. Therefore, our data suggest that accumulation of endog- enous AEA during focal ischemic injury may stem from a specific mechanism involving altered endocannabinoid metabolism. To the best of our knowledge, there is no informa- tion on the putative modulation of the endocannabi- noid system by E 2 in the brain under pathophysiologic conditions. Here, we show that acute administration of a pharmacologic dose of E 2 to male rats prevents the increase in AEA levels produced in the striatum by MCAo, an effect that seems to occur through the modulation of both NAPE-PLD and FAAH. In fact, both enzyme activities returned to control values when rats were pretreated with a neuroprotective dose of the hormone. It seems also noteworthy that E 2 reduced CB1 receptor binding in the ischemic striatum, and it is tempting to speculate that this may further contrib- ute to neuroprotection by reducing the ability of endogenous cannabinoids to evoke CB1-mediated responses. Moreover, we report the original observa- tion that E 2 increased FAAH and reduced NAPE- PLD activity via an ER-dependent mechanism in the ischemic striatum, thus reversing the effects of ischemia on these enzymatic activities. Transient MCAo has been associated with blood–brain barrier disruption [54,55], and under these experimental conditions the antiestrogen ICI182 780 has been shown to reach the brain after systemic administration [56,57]. Thus, it is plausible that the drug is able to cross the blood–brain barrier under our experimental conditions. Collectively, our study demonstrates that focal brain ischemia produced by transient MCAo results in a sig- nificant modulation of the endocannabinoid system, which occurs as early as 2 h following injury and con- tinues during the early stages of reperfusion in the ischemic striatum. Striatal downregulation of FAAH and upregulation of NAPE-PLD activity lead to increased levels of AEA, which in turn may play a role in the pathophysiology of damage occurring in the ischemic brain. More interestingly, we found that the putative neurotoxic effects produced by the MCAo- induced increase of endogenous AEA levels may be significantly blocked by estrogen, possibly through an ER-dependent mechanism. In conclusion, this is the first report documenting the modulation of the endo- cannabinoid system by estrogen in the brain under pathologic conditions, leading to the suggestion that it might be pivotal in hormone-mediated neuroprotection after ischemic stroke. Endocannabinoid system modulation by E 2 after MCAo D. Amantea et al. 4470 FEBS Journal 274 (2007) 4464–4475 ª 2007 The Authors Journal compilation ª 2007 FEBS Experimental procedures Materials Chemicals were of the purest analytical grade. AEA, resinferatoxin (RTX), E 2 and R-(+)-WIN55,212-2 were obtained from Sigma Chemical Co. (St Louis, MO). ICI182 780 was purchased from Tocris Bioscience (Avonmouth, UK). [ 3 H]AEA (223 CiÆmmol )1 ), [ 3 H]RTX (43 CiÆmmol )1 ) and [ 3 H]CP55.940 (5-(1,1¢-dimethylheptyl)- 2-[1R,5R-hydroxy-2R-(3-hydroxypropyl) cyclohexyl]-phenol, 126 Ci mmol )1 ) were purchased from Perkin Elmer Life Sciences (Boston, MA). N-[ 3 H]Arachidonoyl-phosphatidyl- ethanolamine (200 CiÆmmol )1 ) was obtained from ARC (St Louis, MO). N-piperidino-5-(4-chlorophenyl)-1-(2,4-di- chlorophenyl)-4-methyl-3-pyrazole carboxamide (SR141716) was a kind gift of Sanofi-Aventis Recherche (Montpellier, France). Rabbit polyclonal antibodies to CB1R were obtained from Cayman Chemicals (Ann Arbor, MI), rabbit polyclonal antibodies to FAAH [53] were prepared by Primm S.r.l. (Milan, Italy), and goat anti-(rabbit alkaline phosphatase) conjugates (GAR-AP) were obtained from Bio-Rad Laboratories (Hercules, CA). Animals and drug treatments Adult male Wistar rats were purchased from Charles River, Calco, Italy. Animals were housed under controlled envi- ronmental conditions with an ambient temperature of 22 °C, a relative humidity of 65%, and a 12 h light : 12 h dark cycle, with free access to food and water. E 2 was dis- solved in vegetable oil and administered intraperitoneally, 1 h prior to MCAo, at a dose of 0.20 mgÆkg )1 . ICI182 780 was dissolved in 4% dimethylsulfoxide in vegetable oil and administered intraperitoneally at a dose of 0.25 mgÆkg )1 , 1 h before E 2 . SR141716 was dissolved in vegetable oil and administered intraperitoneally at a dose of 3 mgÆkg )1 , 15 min prior to MCAo. R-(+)-WIN55,212-2 was dissolved in propylene glycol and administered intraperitoneally at a dose of 1 mgÆkg )1 , 15 min prior to MCAo. Control rats received a vehicle in which the corresponding drug had been dissolved and that was administered under the same injection schedule as the drug treatment. All the experimental procedures were performed in accor- dance with the guidelines of the European Community Council Directive 86⁄ 609, included in D.M. 116 ⁄ 1992 of the Italian Ministry of Health. Focal cerebral ischemia Brain ischemia was induced by MCAo in male Wistar rats (280–320 g) by intraluminal filament, using the relatively noninvasive technique previously described by Longa et al. [58]. Briefly, rats were anesthetized with 5% isoflurane in air, and were maintained with the lowest acceptable concen- tration of the anesthetic (1.5–2%). Body temperature was measured with a rectal probe and was kept at 37 °C during the surgical procedure with a heating pad. Under an oper- ating microscope, the external and internal right carotid arteries were exposed through a neck incision. The external carotid artery was cut approximately 3 mm above the com- mon carotid artery bifurcation, and a silk suture was tied loosely around the external carotid stump. A silicone- coated nylon filament (diameter: 0.28 mm) was then inserted into the external carotid artery and gently advanced into the internal carotid artery, approximately 18 mm from the carotid bifurcation, until mild resistance was felt, thereby indicating occlusion of the origin of the MCA in the Willis circle. The silk suture was tightened around the intraluminal filament to prevent bleeding. The wound was then sutured and anesthesia discontinued. Sham rats were exposed to the same surgical procedure without occlusion of the MCA. One hour after surgery, the animals were grossly assessed for neurologic deficit as follows: 0 ¼ no deficit, 1 ¼ failure to extend left forelimb, 2 ¼ decreased resistance to lateral push, 3 ¼ circling to contralateral side, 4 ¼ walks only when stimulated, and 5 ¼ no spontaneous motor activity. Only rats with clear neurologic deficits (‡ 3), indicating successful occlusion of the MCA [59], were included in the study. To allow reperfusion, rats were briefly reanesthetized with isoflurane, and the nylon filament was withdrawn 2 h after MCAo. After the discontinuation of isoflurane and wound closure, the animals were allowed to wake and were kept in their cages with free access to food and water. Neuropathology and quantification of ischemic damage Cerebral infarct volume was evaluated 22 h after reperfu- sion in rats subjected to 2 h of MCAo. Rats were killed by decapitation, and the brains were rapidly removed. Eight serial sections from each brain were cut at 2 mm intervals from the frontal pole using a rat brain matrix. To measure ischemic damage, brain slices were stained in a solution containing 2% 2,3,5-triphenyltetrazolium chloride (TTC) in saline, at 37 °C. After 10 min of incubation, the slices were transferred to 10% neutral buffered formaldehyde and stored at 4 °C prior to analysis. Images of TTC-stained sec- tions were captured using a digital scanner and analyzed using image analysis software (imagej, version 1.30). The infarct volume (mm 3 ) was calculated by summing the infarcted area (unstained) of the eight sections and multi- plying by the interval thickness between sections [60]. Analysis of the endocannabinoid system For analysis of the endocannabionoid system, rats were killed by decapitation at different times following MCAo, as indicated; the brains were rapidly dissected out, and D. Amantea et al. Endocannabinoid system modulation by E 2 after MCAo FEBS Journal 274 (2007) 4464–4475 ª 2007 The Authors Journal compilation ª 2007 FEBS 4471 ipsilateral cortical and striatal samples were frozen in liquid nitrogen. For the evaluation of endogenous levels of AEA, rat brain samples were homogenized with an UltraTurrax T25 (Stauffen, Germany) in 50 mm Tris ⁄ HCl, 1 mm EDTA (pH 7.4) and 1 mm phenylmethanesulfonyl fluoride buffer, at a 1 : 10 (w ⁄ v) homogenization ratio. Lipids were then extracted [61], the organic phase was dried under nitrogen, and the dry pellet was derivatized as previously reported [62]. Briefly, 25 lLof10mm 4-(N- chloroformylmethyl-N-methyl)amino-7-N,N-dimethyl-amino- sulfonyl-2,1,3-benzoxadiazole (Tokyo Kasei Kogyo Co., Ltd, Tokyo, Japan) was added to 500 lL anhydrous dichloromethane. The mixture was then heated at 60 °C for 1 h, dried in a centrifugal concentrator (Martin Christ GmbH, Osterode am Hartz, Germany), and reconstituted in 50 lL of acetonitrile. HPLC with fluorimetric detection was carried out using an S-200 fluorescence detector (Perkin-Elmer Life Sciences). The separation was per- formed with a mobile phase of acetonitrile ⁄ water (70 : 30, v ⁄ v) at a flow rate of 1.0 mLÆmin )1 . The concentration of AEA was quantified by comparison with known amounts of standard, as previously reported [61]. The hydrolysis of [ 3 H]AEA by FAAH (EC 3.5.1.4) was measured in rat brain areas (20 lg per test) by RP- HPLC, using 10 lm [ 3 H]AEA, as previously reported [63]. FAAH activity was expressed as pmol arachidonate releasedÆmin )1 Æ(mg protein) )1 . The synthesis of AEA through the activity of NAPE-PLD (EC 3.1.4.4) was assayed in brain homogenates (50 lg per test 1 ), using 100 lm N-[ 3 H]Arachi- donoyl-phosphatidylethanolamine, as previously reported [64]. NAPE-PLD activity was expressed as pmol AEA releasedÆmin )1 Æ(mg protein) )1 . It should be mentioned that a novel biosynthetic pathway for AEA has been recently reported in mouse brain and RAW264.7 macrophages [65]. This pathway involves the phospholipase C-catalyzed cleav- age of NAPE to generate a phosphoanandamide, which is subsequently dephosphorylated by phosphatases. Therefore, NAPE hydrolysis assayed in this study may not be the only mechanism responsible for the production of AEA. The binding of 400 pm [ 3 H]CP55.940 to rat brain membranes was determined through rapid filtration assays [63], and was expressed as fmol CP55.940 boundÆ(mg protein) )1 . Also, the binding of 200 pm [ 3 H]RTX was evaluated by rapid filtration assays, performed as previously reported [66], and was expressed as fmol RTX boundÆ(mg protein) )1 . For both agonists, the binding specificity was checked in the presence of 1 lm ‘cold’ ligand [63,66]. The protein content of CB1 receptors and of FAAH was quantified by ELISA, performed on brain homogenates (20 lg per well) with polyclonal antibodies to CB1 receptor (diluted 1 : 250) or FAAH (1 : 500) [63]. Goat anti-(rabbit alkaline phosphatase) conjugate (diluted 1 : 2000) was used as second antibody, and nonimmune rabbit serum (Primm S.r.l) was used as a control for specificity. Statistical analysis Data are reported as means ± SD or means ± SEM, as indicated. Statistical analysis was performed by the non- parametric Mann–Whitney U -test, or by the unpaired Stu- dent’s t-test (between two groups) or anova (for more than two experimental groups), as indicated. Experimental data were elaborated by means of the instat 3 program or the prism 3 program (GraphPAD Software for Science, San Diego, CA), and differences were considered statistically significant when P < 0.05. Acknowledgements We wish to thank Drs Valeria Gasperi, Chiara De Simone (University of Rome ‘Tor Vergata’), Natalia Battista and Nicoletta Pasquariello (University of Teramo) for their expert assistance with biochemical analysis. 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Modulation of the endocannabinoid system by focal brain ischemia in the rat is involved in neuroprotection afforded by 17b-estradiol Diana. protect the brain against focal ischemia [35]. In order to investigate the role of the endocannabinoid system in the neuroprotection afforded by estrogen, the