glyceryl-2-arachidonate and Brain-Ischemia

Stroke is a major cause of morbidity and mortality and follows heart disease and cancer as the third leading cause of death in Western societies [1]. Despite many advances in stroke research and pharmacotherapy, clinical treatment of this debilitating disorder is still inadequate. Recent findings from several laboratories have identified the endocannabinoid signaling pathway, comprised of the endocannabinoid agonist anandamide and its pharmacological targets, CB1 and CB2 cannabinoid receptors and associated anandamide receptors, as a physiological system with capacity to mitigate cardiovascular and cerebrovascular disorders through neuronal and endothelial actions. Variability in experimental stroke models and modes of outcome evaluation, however, have provoked controversy regarding the precise roles of endocannabinoid signals in mediating neural and/or vascular protection versus neurovascular damage. Clinical trials of the CB1 antagonist rimonabant demonstrate that modulation of endocannabinoid signaling during metabolic regulation of vascular disorders can significantly impact clinical outcomes, thus providing strong argument for therapeutic utility of endocannabinoids and/or cannabinoid receptors as targets for therapeutic intervention in cases of stroke and associated vascular disorders. The purpose of this review is to provide updated information from basic science and clinical perspectives on endocannabinoid ligands and their effects in the pathophysiologic genesis of stroke. Particular emphasis will be placed on the endocannabinoids anandamide and 2-arachidonylglycerol and CB1 receptor-mediated mechanisms in the neurovascular unit during stroke pathogenesis. Deficiencies in our knowledge of endocannabinoids in the etiology and pathogenesis of stroke, caveats and limitations of existing studies, and future directions for investigation will be addressed.

Topics: Arachidonic Acids; Brain Ischemia; Cannabinoid Receptor Modulators; Endocannabinoids; Glycerides; Humans; Matrix Metalloproteinases; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Signal Transduction; Stroke

Hypoxia caused by ischemia induces acidosis and neuroexcitotoxicity, resulting in neuronal death in the central nervous system (CNS). Monoacylglycerol lipase (MAGL) is a modulator of 2-arachidonoylglycerol (2-AG), which is involved in retrograde inhibition of glutamate release in the endocannabinoid system. In the present study, we used positron emission tomography (PET) to monitor MAGL-positive neurons and neuroinflammation in the brains of ischemic rats. Additionally, we performed PET imaging to evaluate the neuroprotective effects of an MAGL inhibitor in an ischemic injury model.

Topics: Animals; Arachidonic Acids; Benzodioxoles; Brain; Brain Ischemia; Carbon Radioisotopes; Cell Hypoxia; Disease Models, Animal; Endocannabinoids; Glycerides; Infarction, Middle Cerebral Artery; Ischemic Stroke; Male; Minocycline; Monoacylglycerol Lipases; Neuroprotective Agents; Piperidines; Positron-Emission Tomography; Rats; Rats, Sprague-Dawley; Stroke; Tomography, X-Ray Computed

Topics: Animals; Arachidonic Acids; Brain Ischemia; Cannabinoids; Cerebrovascular Circulation; Endocannabinoids; Glycerides; Platelet Aggregation; Rats; Stroke

The transient global cerebral hypoperfusion/reperfusion achieved by induction of Bilateral Common Carotid Artery Occlusion followed by Reperfusion (BCCAO/R) may trigger a physiological response in an attempt to preserve tissue and function integrity. There are several candidate molecules among which the endocannabinoid system (ECS) and/or peroxisome-proliferator activated receptor-alpha (PPAR-alpha) may play a role in modulating oxidative stress and inflammation. The aims of the present study are to evaluate whether the ECS, the enzyme cyclooxygenase-2 (COX-2) and PPAR-alpha are involved during BCCAO/R in rat brain, and to identify possible markers of the ongoing BCCAO/R-induced challenge in plasma.. Adult Wistar rats underwent BCCAO/R with 30 min hypoperfusion followed by 60 min reperfusion. The frontal and temporal-occipital cortices and plasma were analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS) to determine concentrations of endocannabinoids (eCBs) and related molecules behaving as ligands of PPAR-alpha, and of oxidative-stress markers such as lipoperoxides, while Western Blot and immunohistochemistry were used to study protein expression of cannabinoid receptors, COX-2 and PPAR-alpha. Unpaired Student's t-test was used to evaluate statistical differences between groups.. The acute BCCAO/R procedure is followed by increased brain tissue levels of the eCBs 2-arachidonoylglycerol and anandamide, palmitoylethanolamide, an avid ligand of PPAR-alpha, lipoperoxides, type 1 (CB1) and type 2 (CB2) cannabinoid receptors, and COX-2, and decreased brain tissue concentrations of docosahexaenoic acid (DHA), one of the major targets of lipid peroxidation. In plasma, increased levels of anandamide and lipoperoxides were observed.. The BCCAO/R stimulated early molecular changes that can be easily traced in brain tissue and plasma, and that are indicative of the tissue physiological response to the reperfusion-induced oxidative stress and inflammation. The observed variations suggest that the positive modulation of the ECS and the increase of proinflammatory substances are directly correlated events. Increase of plasmatic levels of anandamide and lipoperoxides further suggests that dysregulation of these molecules may be taken as an indicator of an ongoing hypoperfusion/reperfusion challenge.

Topics: Amides; Animals; Arachidonic Acids; Brain Ischemia; Carotid Artery, Common; Cerebrovascular Disorders; Cyclooxygenase 2; Docosahexaenoic Acids; Endocannabinoids; Ethanolamines; Frontal Lobe; Gene Expression Regulation; Glycerides; Lipid Peroxidation; Lipid Peroxides; Male; Occipital Lobe; Oxidative Stress; Palmitic Acids; Polyunsaturated Alkamides; PPAR alpha; Rats; Rats, Wistar; Reperfusion Injury; Temporal Lobe

The human N-Myc downstream-regulated gene 2 (NDRG2) is expressed in astrocytes, and may be involved in the modulation of gliacyte function in the central nervous system. Our previous study found suppression of NDRG2 up-regulation in reactive astrocytes in cerebral ischemic tolerance. 2-Arachidonylglycerol (2-AG) can induce cerebral ischemic tolerance. However, the underlying mechanism of NDRG2 in cytoprotection induced by 2-AG in primary astrocytesis still unknown. In this study, we investigated the role of NDRG2 in cerebral ischemic tolerance induced by 2-AG after oxygen-glucose deprivation (OGD) in primary astrocytes. The results showed that primary astrocytes exposed to OGD resulted in marked increase of lactate dehydrogenase (LDH) release and decrease of methyl thiazolyl tetrazolium (MTT) reduction activity in comparison to control cultures. The levels of NDRG2 and phospho-signal transducer and activator of transcription 3 (pSTAT3) in the OGD group were comparably higher than those in the control group, and the up-regulation of NDRG2 and pSTAT3 was suppressed in NDRG2 siRNA group. The cell viability in the 2-AG group was higher than that in the OGD group, and transfecting the NDRG2 pSRL-CDH1-GFP vector reversed the protective effects of 2-AG. The levels of NDRG2 and pSTAT3 in the 2-AG group were lower than those in the OGD group. 2-AG suppressed STAT3 phosphorylation by decreased expression of NDRG2. In conclusion, 2-AG protects primary astrocytes exposed to oxygen-glucose deprivation through a blockade of NDRG2 signaling and STAT3 phosphorylation. These findings bring insight to the roles of NDRG2 in ischemic-hypoxic injury and provide novel potential targets for future potent clinical therapies on cerebral ischemia injury.

Topics: Animals; Animals, Newborn; Arachidonic Acids; Astrocytes; Brain Ischemia; Cell Hypoxia; Cell Survival; Cells, Cultured; Cytoprotection; Dose-Response Relationship, Drug; Down-Regulation; Endocannabinoids; Glucose; Glycerides; Nerve Tissue Proteins; Phosphorylation; Primary Cell Culture; Protective Agents; Rats, Sprague-Dawley; RNA Interference; Signal Transduction; STAT3 Transcription Factor; Transfection

The involvement of brain 2-arachidonoylglycerol (2-AG) in a number of critical physiological and pathophysiological regulatory mechanisms highlights the importance for an accurate brain 2-AG determination. In the present study, we validated head-focused microwave irradiation (MW) as a method to prevent postmortem brain 2-AG alterations before analysis. We compared MW to freezing to prevent 2-AG induction and estimated exogenous and endogenous 2-AG stability upon exposure to MW. Using MW, we measured, for the first time, true 2-AG brain levels under basal conditions, 30 s after brain removal from the cranium, and upon exposure to 5 min of brain global ischemia. Our data indicate that brain 2-AG levels are instantaneously and dramatically increased approximately 60-fold upon brain removal from the cranium. With 5 min of brain global ischemia 2-AG levels are also, but less dramatically, increased 3.5-fold. Our data indicate that brain tissue fixation with MW is a required technique to measure both true basal 2-AG levels and 2-AG alterations under different experimental conditions including global ischemia, and 2-AG is stable upon exposure to MW.

Topics: Animals; Arachidonic Acids; Brain; Brain Ischemia; Endocannabinoids; Glycerides; Microwaves; Rats; Rats, Sprague-Dawley; Tissue Fixation; Up-Regulation

We investigated whether glutamate receptor subunit 2 (GluR2) is involved in EA pretreatment-induced neuroprotection via cannabinoid CB1 receptors (CB1R) after global cerebral ischemia in mice. Two hours after electric acupuncture (EA) pretreatment, global cerebral ischemia (GCI) was induced by bilateral common carotid artery occlusion (BCCAO) for 20 min. The GluR2 expression was examined in the hippocampus after reperfusion. Cell survival, neuronal apoptosis, the Bax/Bcl-2 ratio and neurological scores were evaluated at 24 h after BCCAO in the presence or absence of the GluR2 inhibitor. Furthermore, the GluR2 was determined in the presence and absence of CB1R inhibitor. Our results showed EA pretreatment enhanced expression of GluR2 in the hippocampus 2 h after reperfusion. Moreover, EA pretreatment improved neurological outcome, promoted cell survival, inhibited neuronal apoptosis, and decreased the Bax/Bcl-2 ratio after reperfusion. GluR2 knockdown by GluR2 siRNA effectively reversed the beneficial effects of EA pretreatment. Furthermore, CB1R siRNA and two CB1R antagonists blocked the elevation of GluR2 expression by EA pretreatment, whereas the two CB1R agonists up-regulated GluR2 expression as EA pretreatment. In conclusion, GluR2 up-regulation is involved in neuroprotection of EA pretreatment against GCI through CB1R, suggesting that GluR2 may be a novel target for stroke intervention.

Topics: Animals; Apoptosis; Arachidonic Acids; Brain Ischemia; Cell Survival; Disease Models, Animal; Down-Regulation; Electroacupuncture; Endocannabinoids; Gene Expression Regulation; Gene Knockdown Techniques; Glycerides; Hippocampus; Mice; Pyramidal Cells; Receptor, Cannabinoid, CB1; Receptors, AMPA; Reperfusion; RNA Interference; Time Factors; Up-Regulation

Growing evidence suggests that therapeutic strategies to modulate the post-ischemic inflammatory responses are promising approaches to improve stroke outcome. Although the endocannabinoid system has been emerged as an endogenous therapeutic target to regulate inflammation after stroke insult, the downstream mechanisms and their potentials for therapeutic intervention remain controversial. Here we identified trans- and cis-hinokiresinols as novel non-selective antagonists for two G-protein-coupled cannabinoid receptors, cannabinoid receptor type 1 and type 2. The Electric Cell-substrate Impedance Sensing and Boyden chamber migration assays using primary microglial cultures revealed that both hinokiresinols significantly inhibited an endocannabinoid, 2-arachidonoylglycerol-induced migration. Hinokiresinols modulated 2-arachidonoylglycerol-induced mitochondrial bioenergetics in microglia as evidenced by inhibition of ATP turnover and reduction in respiratory capacity, thereby resulting in impaired migration activity. In rats subjected to transient middle cerebral artery occlusion (1.5-h) followed by 24-h reperfusion, post-ischemic treatment with hinokiresinols (2 and 7-h after the onset of ischemia, 10 mg/kg) significantly reduced cerebral infarct and infiltration of ED1-positive microglial/macrophage cells into cerebral ischemic lesions in vivo. Co-administration of exogenous 2-AG (1 mg/kg, i.v., single dose at 2 h after starting MCAO) abolished the protective effect of trans-hinokiresionol. These results suggest that hinokiresinols may serve as stroke treatment by targeting the endocannabinoid system. Alteration of mitochondrial bioenergetics and consequent inhibition of inflammatory cells migration may be a novel mechanism underlying anti-ischemic effects conferred by cannabinoid receptor antagonists.

Topics: Animals; Arachidonic Acids; Brain Ischemia; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cell Movement; Cell Respiration; Cells, Cultured; Disease Models, Animal; Endocannabinoids; Glycerides; Lignans; Macrophages; Male; Microglia; Mitochondria; Phenols; Rats

The N-acylethanolamines (NAEs) and 2-arachidonoylglycerol (2-AG) are bioactive lipids that can modulate inflammatory responses and protect neurons against glutamatergic excitotoxicity. We have used a model of focal cerebral ischemia in young adult mice to investigate the relationship between focal cerebral ischemia and endogenous NAEs. Over the first 24 h after induction of permanent middle cerebral artery occlusion, we observed a time-dependent increase in all the investigated NAEs, except for anandamide. Moreover, we found an accumulation of 2-AG at 4 h that returned to basal level 12 h after induction of ischemia. Accumulation of NAEs did not depend on regulation of N-acylphosphatidylethanolamine-hydrolyzing phospholipase D or fatty acid amide hydrolase. Treatment with the fatty acid amide hydrolase inhibitor URB597 (cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester; 1 mg/kg; i.p.) 1.5 h before arterial occlusion decreased the infarct volume in our model system. Our results suggest that NAEs and 2-AG may be involved in regulation of neuroprotection during focal cerebral ischemia in mice.

Topics: Analysis of Variance; Animals; Arachidonic Acids; Benzamides; Brain; Brain Infarction; Brain Ischemia; Carbamates; Disease Models, Animal; Endocannabinoids; Enzyme Inhibitors; Ethanolamines; Glycerides; Male; Mice; RNA, Messenger; Time Factors

Endocannabinoids act as neuroprotective molecules promptly released in response to pathological stimuli. Hence, they may represent one component of protection and/or repair mechanisms mobilized by dopamine (DA) neurons under ischemia. Here, we show that the endocannabinoid 2-arachidonoyl-glycerol (2-AG) plays a key role in protecting DA neurons from ischemia-induced altered spontaneous activity both in vitro and in vivo. Accordingly, neuroprotection can be elicited through moderate cannabinoid receptor type-1 (CB1) activation. Conversely, blockade of endocannabinoid actions through CB1 receptor antagonism worsens the outcome of transient ischemia on DA neuronal activity. These findings indicate that 2-AG mediates neuroprotective actions by delaying damage and/or restoring function of DA cells through activation of presynaptic CB1 receptors. Lastly, they point to CB1 receptors as valuable targets in protection of DA neurons against ischemic injury and emphasize the need for a better understanding of endocannabinoid actions in the fine control of DA transmission.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzoxazines; Brain Ischemia; Cannabinoid Receptor Modulators; Dopamine; Electrophysiology; Endocannabinoids; Glycerides; In Vitro Techniques; Male; Mice; Mice, Knockout; Morpholines; Naphthalenes; Neurons; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Rimonabant; Signal Transduction; Ventral Tegmental Area

Vascular endothelial cells are important not only for maintaining homeostasis, but also in pathogenesis of vascular disorders. Cerebral capillary and microvascular endothelial cells play an active role in maintaining cerebral blood flow, microvascular tone and blood brain barrier functions. Factors produced and released by endothelial cells, other brain cells and circulating blood cells participate in these regulatory functions. In particular, endothelin-1 (ET-1) and nitric oxide (NO) are known to contribute to the functional vascular changes under pathological conditions (e.g., hypertension, arteriosclerosis, and stroke). This report describes the involvement of endothelial cell mediators in the post-ischemic hypoperfusion induced by brain ischemia and in vitro endothelial responses (Ca(2+) mobilization and cytoskeletal rearrangements) to ET-1 and its interactions with NO or 2-AG. The capacity of NO and endocannabinoids to counteract ET-1-induced cerebral capillary and microvascular endothelial responses indicates that they may actively participate in EC function and implicates them in physiological and pathophysiological conditions.

Topics: Actins; Analysis of Variance; Animals; Arachidonic Acids; Blood-Brain Barrier; Brain; Brain Ischemia; Calcium; Cells, Cultured; Cytoskeleton; Endocannabinoids; Endothelin-1; Endothelium, Vascular; Gerbillinae; Glycerides; Humans; Immunohistochemistry; Microcirculation; Nitric Oxide; Reperfusion Injury

Focal cerebral ischemia (FCI) induces rapid neuronal death in the ischemic core, which gradually expands toward the penumbra, partly as the result of a neuroinflammatory response. It is known that propagation of neuroinflammation involves microglial cells, the resident macrophages of the brain, which are highly motile when activated by specific signals. However, the signals that increase microglial cell motility in response to FCI remain mostly elusive. Here, we tested the hypothesis that endocannabinoids mediate neuroinflammation propagation by increasing microglial cell motility. We found that, in mouse cerebral cortex, FCI greatly increases palmitoylethanolamide (PEA), only moderately increases anandamide [arachidonylethanolamide (AEA)], and does not affect 2-arachidonoylglycerol levels. We also found that PEA potentiates AEA-induced microglial cell migration, without affecting other steps of microglial activation, such as proliferation, particle engulfment, and nitric oxide production. This potentiation of microglial cell migration by PEA involves reduction in cAMP levels. In line with this, we provide evidence that PEA acts through Gi/o-coupled receptors. Interestingly, these receptors engaged by PEA are pharmacologically distinct from CB1 and CB2 cannabinoid receptors, as well as from the WIN and abn-CBD (abnormal-cannabidiol) receptors, two recently identified cannabinoid receptors. Our results show that PEA and AEA increase after FCI and synergistically enhance microglial cell motility. Because such a response could participate in the propagation of the FCI-induced neuroinflammation within the CNS, and because PEA is likely to act through its own receptor, a better understanding of the receptor engaged by PEA may help guide the search for improved therapies against neuroinflammation.

Topics: Amides; Animals; Arachidonic Acids; Brain Ischemia; Cannabinoid Receptor Modulators; Cannabinoids; Cell Division; Cell Line; Cell Movement; Cerebral Cortex; Endocannabinoids; Ethanolamines; Fatty Acids, Unsaturated; Glycerides; Heterotrimeric GTP-Binding Proteins; Mice; Microglia; Nitric Oxide; Palmitic Acids; Phagocytosis; Polyunsaturated Alkamides; Receptors, Cannabinoid; Receptors, Drug

Rat brain, frozen in liquid nitrogen immediately after decapitation, contains a substantial amount of 2-arachidonoylglycerol (0.34 nmol/g tissue), an endogenous cannabinoid receptor ligand. The level of 2-arachidonoylglycerol in the brain was rapidly augmented after decapitation, the peak being noted 30 s after decapitation (1.54 nmol/g tissue). Noticeably, there are two phases during the increase in the levels of 2-arachidonoylglycerol: a rapid transient increase and a subsequent gradual sustained increase, suggesting that at least two separate mechanisms are involved in the generation of 2-arachidonoylglycerol in the decapitated brain. Gradual sustained formation was also observed for other monoacylglycerols, (e.g. 2-palmitoylglycerol plus 2-oleoylglycerol and 2-cis-vaccenoylglycerol). Thus, it is important to minimize post-mortem changes to estimate the exact tissue levels of 2-arachidonoylglycerol as well as other monoacylglycerols in the brain.

Topics: Animals; Arachidonic Acids; Brain; Brain Chemistry; Brain Ischemia; Chromatography, High Pressure Liquid; Endocannabinoids; Glycerides; Ligands; Male; Postmortem Changes; Rats; Rats, Wistar; Receptors, Cannabinoid; Receptors, Drug

The endogenous cannabinoids (endocannabinoids) anandamide and 2-arachidonylglycerol increased cell viability in cerebral cortical neuron cultures subjected to 8 h of hypoxia and glucose deprivation. This effect was observed at nanomolar concentrations, was reproduced by a non-hydrolyzable analog of anandamide, and was unaltered by CB1 or CB2 cannabinoid receptor antagonists. Like synthetic cannabinoids, endocannabinoids can protect neurons from hypoxic injury, and may represent endogenous neuroprotective molecules in cerebral ischemia.

Topics: Animals; Arachidonic Acids; Brain Ischemia; Cannabinoid Receptor Modulators; Cannabinoids; Cell Hypoxia; Cell Survival; Cells, Cultured; Cerebral Cortex; Dizocilpine Maleate; Endocannabinoids; Excitatory Amino Acid Antagonists; Glycerides; Neurons; Neuroprotective Agents; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley