anandamide and Reperfusion-Injury

Arachidonoyl ethanolamide (anandamide, AEA) and 2-arachidonoylglycerol (2-AG) are the most studies endocannabinoids. AEA and 2-AG are degraded by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) enzymes, respectively. FAAH and MAGL enzymes are widely expressed in many tissues, including kidney. Recent works have depicted that AEA and 2-AG levels are associated with ischemia-reperfusion (IR) injury. In this study, we investigated the effects of MAGL inhibitor KML29 and FAAH inhibitor URB597 against kidney IR injury.. The kidneys of the rats underwent ischemia for 45 min and then reperfusion for 24 h. KML29 and URB597 were administered intraperitoneally with kidney IR to two different treatment groups.. IR application increased serum blood urea nitrogen (BUN), creatinine (Cre), interleukin-18 (IL-18), neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule-1 (KIM-1) levels, while these parameters were decreased following KML29 and URB597 administration. KML29 and URB597 administration also reduced the increased toll-like receptor-4 (TRL-4), phosphorylated-NF-κB, phosphorylated-IκB-α, tumor necrosis factor alpha (TNF-α), interleukin-1beta (IL-1β), interleukin-6 (IL-6), caspase-3 levels and histopathological damage in kidney tissue.. Our results reveal that MAGL inhibitor KML29 and FAAH inhibitor URB597 have a protective effect on kidney IR injury by preventing apoptosis and inflammation. Inhibition of MAGL and FAAH may be a new therapeutic strategy to prevent kidney IR injury.

Topics: Amidohydrolases; Animals; Endocannabinoids; Kidney; Monoacylglycerol Lipases; Monoglycerides; NF-kappa B; Rats; Reperfusion Injury; Toll-Like Receptor 4

Acute kidney injury (AKI) is a frequent and critical complication in the clinical setting. In rodents, AKI can be effectively prevented through caloric restriction (CR), which has also been shown to increase lifespan in many species. In

Topics: Acute Kidney Injury; Adult; Aged; Animals; Arachidonic Acids; Caenorhabditis elegans; Caloric Restriction; Disease Models, Animal; Endocannabinoids; Female; Humans; Kidney; Longevity; Male; Mice; Mice, Inbred C57BL; Middle Aged; Polyunsaturated Alkamides; Reperfusion Injury

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

Endocannabinoids alter permeability at various epithelial barriers, and cannabinoid receptors and endocannabinoid levels are elevated by stroke, with potential neuroprotective effects. We therefore explored the role of endocannabinoids in modulating blood-brain barrier (BBB) permeability in normal conditions and in an ischaemia/reperfusion model.. Human brain microvascular endothelial cell and astrocyte co-cultures modelled the BBB. Ischaemia was modelled by oxygen-glucose deprivation (OGD) and permeability was measured by transepithelial electrical resistance. Endocannabinoids or endocannabinoid-like compounds were assessed for their ability to modulate baseline permeability or OGD-induced hyperpermeability. Target sites of action were investigated using receptor antagonists and subsequently identified with real-time PCR.. Anandamide (10 μM) and oleoylethanolamide (OEA, 10 μM) decreased BBB permeability (i.e. increased resistance). This was mediated by cannabinoid CB2 receptors, transient receptor potential vanilloid 1 (TRPV1) channels, calcitonin gene-regulated peptide (CGRP) receptor (anandamide only) and PPARα (OEA only). Application of OEA, palmitoylethanolamide (both PPARα mediated) or virodhamine (all 10 μM) decreased the OGD-induced increase in permeability during reperfusion. 2-Arachidonoyl glycerol, noladin ether and oleamide did not affect BBB permeability in normal or OGD conditions. N-arachidonoyl-dopamine increased permeability through a cytotoxic mechanism. PPARα and γ, CB1 receptors, TRPV1 channels and CGRP receptors were expressed in both cell types, but mRNA for CB2 receptors was only present in astrocytes.. The endocannabinoids may play an important modulatory role in normal BBB physiology, and also afford protection to the BBB during ischaemic stroke, through a number of target sites.

Topics: Arachidonic Acids; Astrocytes; Blood-Brain Barrier; Cells, Cultured; Coculture Techniques; Electric Impedance; Endocannabinoids; Endothelial Cells; Humans; Oleic Acids; Permeability; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB2; Reperfusion Injury; RNA, Messenger

Anandamide (AEA), one of endocannabinoids, has been reported to exhibit a cardioprotective ability to limit the damage produced by ischemia-reperfusion injury. AEA reportedly enhanced heat shock protein 72 (HSP72) and HSP25 expression in lungs to protect against lung inflammation. This study tested the hypothesis that intravenously injected AEA would induce HSP72 in the heart and thus render cardioprotection against ischemia-reperfusion injury in rats. Cardiac expression of HSPs was quantitatively evaluated in rats by Western blot analysis. That intravenously injected AEA 1 mg/kg in vivo induced expression of HSP72, which peaked at 24 h after administration. The enhancement of HSP72 by AEA was blocked by cannabinoid 2 (CB(2)) receptor antagonist AM630, but not cannabinoid 1 (CB(1)) receptor antagonist AM251. Therefore, the rats were induced with a 30-min coronary occlusion followed by a 120-min reperfusion in vivo at 24 h after administration of drugs or vehicle, and then the infarct size was measured. AEA reduced myocardial infarct size compared to control group. Pretreatment with AM630 but not AM251 abolished the infarct size-limiting effect of AEA. Further study demonstrated pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, Akt inhibitor MK-2206 and AM630 attenuated phosphorylation of Akt and AEA-induced HSP72 expression. The results suggest that AEA is cardioprotective against ischemia-reperfusion insult through its induction of HSP72, which might be mediated by the PI3K/Akt signaling pathway. These effects were mediated by CB(2) but not CB(1) receptors.

Topics: Animals; Arachidonic Acids; Endocannabinoids; Heart; HSP72 Heat-Shock Proteins; Male; Myocardial Reperfusion Injury; Myocardium; Phosphatidylinositol 3-Kinases; Polyunsaturated Alkamides; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Reperfusion Injury; Signal Transduction

Acute mesenteric ischemia is an entity characterized by rapid developing of circulatory failure. Reperfusion following ischemia causes further mucosal injury.. In our study, an experimental model of 15 minutes of reperfusion following 45 minutes of superior mesenteric artery occlusion was established. The segments which underwent I/R injury were histopathologically examined, and blood samples obtained from the heart were analyzed for alkaline phosphatase and creatine kinase levels.. The results of the study demonstrated that mucosal injury in anandamide injected group was less expressed than in other groups suggesting that anandamide might have a protective effect on the mucosa. After L-NAME and indomethacin injection, the protective effect of anandamide seems to disappear due to inhibition of NO and prostaglandins. The results of histopathological examination of specimens from CB1 receptor and anandamide injected group indicate that I/R injury has regressed.. The protective effect of endogenous anandamide on I/R injury may take place through CB2 receptors in the small intestine; NO and prostaglandin, which are activated through the stimulation of CB2 receptors may be responsible for this protective effect (Fig. 8, Ref. 29). Full Text (Free, PDF) www.bmj.sk.

Topics: Acute Disease; Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Endocannabinoids; Enzyme Inhibitors; Guinea Pigs; Indomethacin; Intestinal Mucosa; Jejunum; Mesentery; NG-Nitroarginine Methyl Ester; Nitric Oxide; Polyunsaturated Alkamides; Reperfusion Injury

Artery occlusion of an organ results in ischemia. When the occlusion is opened and blood flow reinstated there will be tissue injuries identified as reperfusion-induced ischemia (RII). It has been suggested that cannabinoids (CBs) may be involved in the RII. In this study, we assessed the effect of different doses of anandamide analogs and CB receptor agonists: arachidonylcyclopropylamide (ACPA, a CB1 agonist) and JWH133 (a CB2 agonist) in the RII of the mouse kidney. Three doses (0.2, 1 and 5mg/kg, i.p.) of ACPA or JWH133 were used 30min prior initiation of RII. Kidneys were removed 2 and 24h following RII and checked histologically for the grading of ischemic injury. Appropriate control groups were used as well. RII produced lesion comparable with that of ischemia. Different doses of ACPA or JWH133 prevented RII-induced lesions. It is suggestive of the CB system involvement in the kidney RII in mice.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Agonists; Cannabinoids; Endocannabinoids; Female; Kidney; Mice; Polyunsaturated Alkamides; Reperfusion Injury

Hepatic ischemia-reperfusion (I/R) injury continues to be a fatal complication that can follow liver surgery or transplantation. We have investigated the involvement of the endocannabinoid system in hepatic I/R injury using an in vivo mouse model. Here we report that I/R triggers several-fold increases in the hepatic levels of the endocannabinoids anandamide and 2-arachidonoylglycerol, which originate from hepatocytes, Kupffer, and endothelial cells. The I/R-induced increased tissue endocannabinoid levels positively correlate with the degree of hepatic damage and serum TNF-alpha, MIP-1alpha, and MIP-2 levels. Furthermore, a brief exposure of hepatocytes to various oxidants (H2O2 and peroxynitrite) or inflammatory stimuli (endotoxin and TNF-alpha) also increases endocannabinoid levels. Activation of CB2 cannabinoid receptors by JWH133 protects against I/R damage by decreasing inflammatory cell infiltration, tissue and serum TNF-alpha, MIP-1alpha and MIP-2 levels, tissue lipid peroxidation, and expression of adhesion molecule ICAM-1 in vivo. JWH133 also attenuates the TNF-alpha-induced ICAM-1 and VCAM-1 expression in human liver sinusoidal endothelial cells (HLSECs) and the adhesion of human neutrophils to HLSECs in vitro. Consistent with the protective role of CB2 receptor activation, CB2-/- mice develop increased I/R-induced tissue damage and proinflammatory phenotype. These findings suggest that oxidative/nitrosative stress and inflammatory stimuli may trigger endocannabinoid production, and indicate that targeting CB2 cannabinoid receptors may represent a novel protective strategy against I/R injury. We also demonstrate that CB2-/- mice have a normal hemodynamic profile.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Disease Models, Animal; Endocannabinoids; Glycerides; Humans; Inflammation; Liver; Liver Diseases; Mice; Mice, Knockout; Oxidative Stress; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB2; Reperfusion Injury; Up-Regulation

To evaluate whether high intraocular pressure (IOP)-induced ischemia is associated with modifications in the retinal endocannabinoid metabolism and to ascertain whether drugs that interfere with the endocannabinoid system may prevent retinal damage due to ischemic insult.. Anandamide (AEA) synthesis, transport, hydrolysis, and AEA endogenous levels were assessed by means of high-performance liquid chromatography in the retinas of rats undergoing 45 minutes of ischemia followed by 12 hours of reperfusion. Under these experimental conditions, binding to cannabinoid (CB1R) and vanilloid (TRPV1) receptor was assessed with rapid-filtration assays. AEA-hydrolase (FAAH, fatty acid amide hydrolase), CB1R and TRPV1 protein content was determined by enzyme-linked immunosorbent assay. Finally, to characterize the neuroprotective profile of drugs that interfere with the endocannabinoid system, cell counting in the retinal ganglion cell (RGC) layer and real-time polymerase chain reactions for Thy-1 mRNA expression were used.. In rat retina, ischemic insult followed by reperfusion resulted in enhanced FAAH activity and protein expression paralleled by a significant decrease in the endogenous AEA tone, whereas the AEA-membrane transporter or the AEA-synthase NAPE-PLD (N-acyl-phosphatidylethanolamine-hydrolyzing-phospholipase-d) were not affected. Retinal ischemia-reperfusion decreased the expression of cannabinoid (CB1) and vanilloid (TRPV1) receptors. Systemic administration of a specific FAAH inhibitor (e.g., URB597) reduced enzyme activity and minimized the retinal damage observed in ischemic-reperfused samples. Similarly, intravitreal injection of the AEA stable analogue, R(+)-methanandamide, reduced cell loss in the RGC layer, and this was prevented by systemic administration of a CB1 or TRPV1 selective antagonist (e.g., SR141716 and capsazepine, respectively).. The original observation that retinal ischemia-reperfusion reduces endogenous AEA via enhanced expression of FAAH supports the deduction that this is implicated in retinal cell loss caused by high IOP in the RGC layer.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Modulators; Capsaicin; Carbamates; Cell Count; Chromatography, High Pressure Liquid; Endocannabinoids; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Hydrolysis; Intraocular Pressure; Male; Ocular Hypertension; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Reperfusion Injury; Retinal Diseases; Retinal Ganglion Cells; Retinal Vessels; Reverse Transcriptase Polymerase Chain Reaction; Rimonabant; RNA, Messenger; Thy-1 Antigens; TRPV Cation Channels

Although the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide share a similar pharmacology, 2-AG reportedly limits myocardial ischaemia-reperfusion injury whereas anandamide does not. We therefore investigated whether or not anandamide reduces infarct size and which, if any, of the known cannabinoid-signalling pathways are involved. Rat isolated perfused hearts were subjected to global, no-flow ischaemia (30 min) and reperfusion (1 h). Agonists were present from 5 min before ischaemia until the end of reperfusion. Antagonists, where used, were present throughout the protocol. Recovery of left ventricular developed pressure and coronary flow was incomplete in control hearts and not significantly affected by any drug treatment. In vehicle-treated hearts, 26+/-3% (n=13) of the left ventricle was infarcted at the end of reperfusion. Infarction of the left ventricle was significantly reduced after 1 microM anandamide (10+/-1%, n=7) or 1 microM methanandamide (12+/-4%, n=6) but not 1 microM HU210. Neither ACPA (1 microM; CB1 receptor agonist) nor JWH133 (1 microM; CB2 receptor agonist), individually or combined significantly affected infarct size. Anandamide (1 microM) did not reduce infarct size in the presence of the CB1 receptor antagonist rimonabant (SR141716A, 1 microM) or the CB2 receptor antagonist, SR144528 (1 microM). Despite sensitivity to CB1 and CB2 receptor antagonists, the infarct-limiting action of anandamide was not mimicked by agonists selective for CB1 or CB2 receptors suggesting the involvement of a novel cannabinoid site of action.

Topics: Animals; Arachidonic Acids; Blood Pressure; Cannabinoid Receptor Modulators; Coronary Circulation; Dimethyl Sulfoxide; Endocannabinoids; Heart; In Vitro Techniques; Male; Myocardial Infarction; Perfusion; Polyunsaturated Alkamides; Rats; Rats, Wistar; Reperfusion Injury; Ventricular Pressure

The role of endocannabinoid signaling in the response of the brain to injury is tantalizing but not clear. In this study, transient middle cerebral artery occlusion (MCAo) was used to produce ischemia/reperfusion injury. Brain content of N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol were determined during MCAo. Whole brain AEA content was significantly increased after 30, 60 and 120 min MCAo compared with sham-operated brain. The increase in AEA was localized to the ischemic hemisphere after 30 min MCAo, but at 60 and 120 min, was also increased in the contralateral hemisphere. 2-Arachidonoylglycerol content was unaffected by MCAo. In a second set of studies, injury was assessed 24 h after 2 h MCAo. Rats administered a single dose (3 mg/kg) of the cannabinoid receptor type 1 (CB1) receptor antagonist SR141716 prior to MCAo exhibited a 50% reduction in infarct volume and a 40% improvement in neurological function compared with vehicle control. A second CB1 receptor antagonist, LY320135 (6 mg/kg), also significantly improved neurological function. The CB1 receptor agonist, WIN 55212-2 (0.1-1 mg/kg) did not affect either infarct volume or neurological score.

Topics: Animals; Arachidonic Acids; Benzofurans; Benzoxazines; Blood Pressure; Brain Chemistry; Brain Infarction; Chromatography, Liquid; Disease Models, Animal; Dose-Response Relationship, Drug; Endocannabinoids; Hemodynamics; Infarction, Middle Cerebral Artery; Ischemic Attack, Transient; Male; Mass Spectrometry; Morpholines; Naphthalenes; Neurologic Examination; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Reperfusion Injury; Rimonabant; Tetrazolium Salts; Time Factors

Following on from previous studies on dermal inflammation in the isolated perfused bovine udder, a new in vitro model of the isolated haemoperfused bovine uterus was established for studies on acute inflammatory reactions (for example, eicosanoid synthesis and regulation of cyclooxygenase-1 [COX-1] and COX-2) caused by ischaemia-reperfusion (I-R) injury. The organs and blood used in this study were obtained from a slaughterhouse. Within 2 hours of slaughter, uterine perfusion was re-established, by using a mixture of homologous blood and Tyrode solution (4:1). After equilibration, several deposits of arachidonic acid (5 mg and 0.1 mg) and arachidonylethanolamide (0.1 mg) were injected into the myometrial tissue. Tissue biopsies were taken from treated and untreated areas at 180 and 300 minutes after the onset of haemoperfusion, for measuring prostaglandin E(2) (PGE(2)) levels. In addition, the regulation of COX-1 and COX-2 mRNA was investigated by using the reverse transcriptase-polymerase chain reaction. Eicosanoid levels were determined by using an enzyme immunoassay (ELISA). Because both an increase in PGE(2) concentration and up-regulation of COX mRNA were observed, the inhibitory effects of dexamethasone, added to the perfusion medium, were studied. Dexamethasone caused a significant decrease in tissue PGE(2) production, but did not induce down-regulation of COX-2 mRNA. In conclusion, the isolated haemoperfused bovine uterus was introduced as an in vitro model of acute inflammation, induced by I-R injury. The suitability of the model for investigating anti-inflammatory substances was demonstrated. Use of the isolated haemoperfused bovine uterus in pharmacological research and drug screening may contribute to reducing the number of animals used for testing.

Topics: Animal Testing Alternatives; Animals; Arachidonic Acid; Arachidonic Acids; Cattle; Cyclooxygenase 2; Dexamethasone; Dinoprostone; Dose-Response Relationship, Drug; Endocannabinoids; Female; Gene Expression Regulation; Hemoperfusion; Inflammation; Isoenzymes; Myometrium; Organ Culture Techniques; Polyunsaturated Alkamides; Prostaglandin-Endoperoxide Synthases; Reperfusion Injury; Up-Regulation; Uterus