Involvement of the endocannabinoid system in attentional modulation of nociceptive behaviour in rats.

BACKGROUND: Distraction is used clinically to relieve and manage pain. It is hypothesized that pain demands attention and that exposure to another attention-demanding stimulus causes withdrawal of attention away from painful stimuli, thereby reducing perceived pain. We have recently developed a rat model that provides an opportunity to investigate the neurobiological mechanisms mediating distraction-induced analgesia, as these mechanisms are, at present, poorly understood. Given the well-described role of the endogenous cannabinoid (endocannabinoid; EC) system in the modulation of pain and attentional processing, the present study investigated its role in distraction-induced antinociception in rats. METHODS: Animals received the CB1 receptor antagonist/inverse agonist, rimonabant or vehicle intraperitoneally, 30 min prior to behavioural evaluation. Formalin-evoked nociceptive behaviour was measured in the presence or absence of a novel-object distractor. Liquid chromatography-tandem mass spectrometry was used to determine the levels of the endogenous cannabinoids anandamide and 2-arachidonoylglycerol (2-AG) in the ventral hippocampus (vHip). RESULTS: Exposure to a novel object distractor significantly reduced formalin-evoked nociceptive behaviour. The novel object-induced reduction in nociceptive behaviour was attenuated by rimonabant. Novel object exposure was also associated with increased tissue levels of anandamide and 2-AG in the vHip. CONCLUSIONS: These data suggest that the reduction in formalin-evoked nociceptive behaviour that occurs as a result of exposure to a novel object may be mediated by engagement of the EC system, in particular in the vHip. The results provide evidence that the EC system may be an important neural substrate subserving attentional modulation of pain.

The monoacylglycerol lipase inhibitor JZL184 attenuates LPS-induced increases in cytokine expression in the rat frontal cortex and plasma: differential mechanisms of action.

BACKGROUND AND PURPOSE: JZL184 is a selective inhibitor of monoacylglycerol lipase (MAGL), the enzyme that preferentially catabolizes the endocannabinoid 2-arachidonoyl glycerol (2-AG). Here, we have studied the effects of JZL184 on inflammatory cytokines in the brain and plasma following an acute immune challenge and the underlying receptor and molecular mechanisms involved. EXPERIMENTAL APPROACH: JZL184 and/or the CB1 receptor antagonist, AM251 or the CB2 receptor antagonist, AM630 were administered to rats 30 min before lipopolysaccharide (LPS). 2 h later cytokine expression and levels, MAGL activity, 2-AG, arachidonic acid and prostaglandin levels were measured in the frontal cortex, plasma and spleen. KEY RESULTS: JZL184 attenuated LPS-induced increases in IL-1ß, IL-6, TNF-α and IL-10 but not the expression of the inhibitor of NFkB (IκBα) in rat frontal cortex. AM251 attenuated JZL184-induced decreases in frontal cortical IL-1ß expression. Although arachidonic acid levels in the frontal cortex were reduced in JZL184-treated rats, MAGL activity, 2-AG, PGE2 and PGD2 were unchanged. In comparison, MAGL activity was inhibited and 2-AG levels enhanced in the spleen following JZL184. In plasma, LPS-induced increases in TNF-α and IL-10 levels were attenuated by JZL184, an effect partially blocked by AM251. In addition, AM630 blocked LPS-induced increases in plasma IL-1ß in the presence, but not absence, of JZL184. CONCLUSION AND IMPLICATIONS: Inhibition of peripheral MAGL in rats by JZL184 suppressed LPS-induced circulating cytokines that in turn may modulate central cytokine expression. The data provide further evidence for the endocannabinoid system as a therapeutic target in treatment of central and peripheral inflammatory disorders.

The endocannabinoid system in the rat dorsolateral periaqueductal grey mediates fear-conditioned analgesia and controls fear expression in the presence of nociceptive tone.

BACKGROUND AND PURPOSE: Endocannabinoids in the midbrain periaqueductal grey (PAG) modulate nociception and unconditioned stress-induced analgesia; however, their role in fear-conditioned analgesia (FCA) has not been examined. The present study examined the role of the endocannabinoid system in the dorsolateral (dl) PAG in formalin-evoked nociceptive behaviour, conditioned fear and FCA in rats. EXPERIMENTAL APPROACH: Rats received intra-dlPAG administration of the CB(1) receptor antagonist/inverse agonist rimonabant, or vehicle, before re-exposure to a context paired 24 h previously with foot shock. Formalin-evoked nociceptive behaviour and fear-related behaviours (freezing and 22 kHz ultrasonic vocalization) were assessed. In a separate cohort, levels of endocannabinoids [2-arachidonoyl glycerol (2-AG) and N-arachidonoyl ethanolamide (anandamide; AEA)] and the related N-acylethanolamines (NAEs) [N-palmitoyl ethanolamide (PEA) and N-oleoyl ethanolamide (OEA)] were measured in dlPAG tissue following re-exposure to conditioned context in the presence or absence of formalin-evoked nociceptive tone. KEY RESULTS: Re-exposure of rats to the context previously associated with foot shock resulted in FCA. Intra-dlPAG administration of rimonabant significantly attenuated FCA and fear-related behaviours expressed in the presence of nociceptive tone. Conditioned fear without formalin-evoked nociceptive tone was associated with increased levels of 2-AG, AEA, PEA and OEA in the dlPAG. FCA was specifically associated with an increase in AEA levels in the dlPAG. CONCLUSIONS AND IMPLICATIONS: Conditioned fear to context mobilises endocannabinoids and NAEs in the dlPAG. These data support a role for endocannabinoids in the dlPAG in mediating the potent suppression of pain responding which occurs during exposure to conditioned aversive contexts. LINKED ARTICLES: This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

Pharmacological inhibition of endocannabinoid degradation modulates the expression of inflammatory mediators in the hypothalamus following an immunological stressor.

The endocannabinoid system is an important regulator of the nervous, neuroendocrine, and immune systems, thus representing a novel therapeutic target for stress-related neuroinflammatory and psychiatric disorders. However, there is a paucity of data relating to the effects of endocannabinoids on neuroinflammatory mediators following an immune stress/challenge in vivo. This study investigated the effects of URB597, a selective inhibitor of fatty acid amide hydrolyase (FAAH), the enzyme that preferentially metabolizes anandamide, on lipopolysaccharide (LPS)-induced increases in the expression of immune mediators in the hypothalamus. Systemic administration of URB597 increased the levels of anandamide and the related N-acylethanolamines, N-palmitoylethanolamide, and N-oleoylethanolamide, but not 2-arachidonoyl glycerol, in the hypothalamus and spleen. URB597 attenuated the LPS-induced increase in interleukin (IL)-1ß expression while concurrently augmenting the LPS-induced increase in suppressor of cytokine signalling (SOCS)-3 expression. In addition, URB597 tended to enhance and reduce the LPS-induced increase in IL-6 and IL-10 mRNA expression, respectively. LPS-induced increases in peripheral cytokine levels or plasma corticosterone were not altered by URB597. The present study provides evidence for a role for FAAH in the regulation of LPS-induced expression of inflammatory mediators in the hypothalamus. Improved understanding of endocannabinoid-mediated regulation of neuroimmune function has fundamental physiological and potential therapeutic significance in the context of stress-related disorders.

Optimization of an enzyme immunoassay for 11-dehydro-thromboxane B(2) in urine: comparison with GC-MS.

The urinary excretion of stable metabolites of thromboxane A2, such as 11-dehydro-thromboxane B2, reflects platelet activity in vivo. Efficient sample purification is required before analysis of thromboxane metabolites, due to the presence of large amounts of interfering material in urine. Analysis by gas chromatography-mass spectrometry after extensive sample work-up procedures provides the most reliable data, but detection by enzyme immunoassay may be reliable if sample cleanup is adequate. We describe an improved immunoassay procedure for 11-dehydro-thromboxane B2, which is based on a simple one-step solid phase extraction, by using Bond-Elut Certify II columns, followed by enzyme immunoassay by using commercially available reagents. 11-Dehydro-thromboxane B2 exists in two forms, with different chemical and immunological characteristics, which are in pH-dependent equilibrium. We kept 11-dehydrothromboxane B2 in its open ring form throughout the assay, by incubating and handling samples at pH 8.6. The extraction step achieved a recovery of 83% (95% confidence interval 74-92%), the sensitivity of the enzyme immunoassay was doubled, and the reproducibility of the assay improved under these conditions. Intra- and interassay coefficients of variation were 3 and 13.8%, respectively. A single 500-mg dose of aspirin reduced the excretion of 11-dehydro-thromboxane B2 by 77+/-14%, suggesting good specificity. Comparison with gas chromatography-mass spectrometry in 28 urine samples showed excellent agreement between the two methods (r2 = 0.94; p<0.0001), and a regression line with a slope close to 1.0. The presently modified enzyme immunoassay for 11-dehydro-thromboxane B2 is suitable for clinical studies evaluating platelet function in vivo and has the advantage of being simpler and less expensive to use than gas chromatography-mass spectrometry.