Mariapia Colleoni

The endogenous fatty acid amide, palmitoylethanolamide, has anti-allodynic and anti-hyperalgesic effects in a murine model of neuropathic pain: involvement of CB(1), TRPV1 and PPARgamma receptors and neurotrophic factors.

Abstract Palmitoylethanolamide (PEA) is an endogenous lipid that is thought to be involved in endogenous protective mechanisms activated as a result of stimulation of inflammatory response. In spite of the well demonstrated anti‐inflammatory properties of PEA, its involvement in controlling pain pathways still remains poorly characterized. On this basis, we tested the efficacy of PEA in vivo against a peculiar persistent pain, such as neuropathic one. PEA was administered i.p. to mice with chronic constriction injury of sciatic nerve (CCI) once a day for one week starting the day after the lesion. This therapeutic regimen evoked a relief of both thermal hyperalgesia and mechanical allodynia in neuropathic mice. Various selective receptor antagonists were used in order to clarify the relative contribution of cannabinoid, vanilloid and peroxisome proliferator‐activated receptor to PEA‐induced effects. The results indicated that CB1, PPARγ and TRPV1 receptors mediated the antinociception induced by PEA, suggesting that the most likely mechanism might be the so‐called “entourage effect” due to the PEA‐induced inhibition of the enzyme catalyzing the endocannabinoid anandamide (AEA) degradation that leads to an enhancement of its tissue levels thus increasing its analgesic action. In addition, the hypothesis that PEA might act through the modulation of local mast cells degranulation is sustained by our findings showing that PEA significantly reduced the production of many mediators such as TNFα and neurotrophic factors, like NGF. The findings presented here, in addition to prove the beneficial effects of PEA in chronic pain, identify new potential targets for analgesic medicine.

Antiinflammatory action of endocannabinoid palmitoylethanolamide and the synthetic cannabinoid nabilone in a model of acute inflammation in the rat

The antiinflammatory activity of synthetic cannabinoid nabilone in the rat model of carrageenan‐induced acute hindpaw inflammation was compared with that of the endocannabinoid palmitoylethanolamide and the nonsteroidal antiinflammatory drug indomethacin. Preliminary experiments in rats used a tetrad of behavioural tests, specific for tetrahydrocannabinol‐type activity in the CNS. These showed that the oral dose of nabilone 2.5 mg kg−1 had no cannabinoid psychoactivity. Intraplantar injection of carrageenan (1% w v−1) elicited a time‐dependent increase in paw volume and thermal hyperalgesia. Nabilone (0.75, 1.5, 2.5 mg kg−1, p.o.), given 1 h before carrageenan, reduced the development of oedema and the associated hyperalgesia in a dose‐related manner. Nabilone 2.5 mg kg−1, palmitoylethanolamide 10 mg kg−1 and indomethacin 5 mg kg−1, given p.o. 1 h before carrageenan, also reduced the inflammatory parameters in a time‐dependent manner. The selective CB2 cannabinoid receptor antagonist {N‐[(1S)‐endo‐1,3,3‐trimethyl bicyclo [2.2.1]heptan‐2‐yl]‐5‐(4‐chloro‐3‐methylphenyl)‐1‐(4‐methylbenzyl)pyrazole‐3 carboxamide} (SR 144528), 3 mg kg−1 p.o. 1 h before nabilone and palmitoylethanolamide, prevented the anti‐oedema and antihyperalgesic effects of the two cannabinoid agonists 3 h after carrageenan. Our findings show the antiinflammatory effect of nabilone and confirm that of palmitoylethanolamide indicating that these actions are mediated by an uncharacterized CB2‐like cannabinoid receptor.

Vanilloid TRPV1 receptor mediates the antihyperalgesic effect of the nonpsychoactive cannabinoid, cannabidiol, in a rat model of acute inflammation

Cannabidiol (CBD), a nonpsychoactive marijuana constituent, was recently shown as an oral antihyperalgesic compound in a rat model of acute inflammation. We examined whether the CBD antihyperalgesic effect could be mediated by cannabinoid receptor type 1 (CB1) or cannabinoid receptor type 2 (CB2) and/or by transient receptor potential vanilloid type 1 (TRPV1). Rats received CBD (10 mg kg−1) and the selective antagonists: SR141716 (N‐(piperidin‐1‐yl)‐5‐(4‐chlorophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide) for CB1, SR144528 (N‐[(1S)‐endo‐1,3,3‐trimethylbicyclo[2.2.1]heptan‐2‐yl]‐5‐(4‐chloro‐3‐methylphenyl)‐1‐(4‐methylbenzyl)pyrazole‐3 carboxamide) for CB2 and capsazepine (CPZ) for TRPV1 receptors. The intraplantar injection of carrageenan in rats induced a time‐dependent thermal hyperalgesia, which peaked at 3 h and decreased at the following times. CBD, administered 2 h after carrageenan, abolished the hyperalgesia to the thermal stimulus evaluated by plantar test. Neither SR141716 (0.5 mg kg−1) nor SR144528 (3 and 10 mg kg−1) modified the CBD‐induced antihyperalgesia; CPZ partially at the lowest dose (2 mg kg−1) and fully at the highest dose (10 mg kg−1) reversed this effect. These results demonstrate that TRPV1 receptor could be a molecular target of the CBD antihyperalgesic action.

The soy isoflavone genistein reverses oxidative and inflammatory state, neuropathic pain, neurotrophic and vasculature deficits in diabetes mouse model.

Treatment of diabetes complications remains a substantial challenge. The aim of this study was to explore the ability of the soy isoflavone genistein in attenuating the signs that follow diabetes onset: nociceptive hypersensitivity, oxidative and inflammatory state, nerve growth factor (NGF) decrease and vascular dysfunctions. Genistein (3 and 6 mg/kg) was administered to C57BL/6J streptozotocin diabetic mice from the 2nd till the 5th week after disease induction. The hind paw withdrawal threshold to mechanical stimulation (tactile allodynia) was evaluated by a von Frey filament. The oxidative stress was assessed measuring: reactive oxygen species by fluorimetric analysis, both the lipoperoxide content, as malondialdehyde, the antioxidant enzymatic activities spectrophotometrically and the glutathione content spectrofluorimetrically. Proinflammatory cytokines and NGF were measured in the sciatic nerve by enzyme-linked immunosorbent assay. Aortic inducible (iNOS) and endothelial nitric oxide synthase (eNOS) protein content was measured by western immunoblotting. Genistein relieved diabetic peripheral painful neuropathy, reverted the proinflammatory cytokine and reactive oxygen species overproduction, and restored the NGF content in diabetic sciatic nerve. Furthermore it restored the GSH content and the GSH and GSSG ratio, improved the antioxidant enzymes activities, decreased reactive oxygen species and lipoperoxide level in the brain and liver. Finally it restored the iNOS and eNOS content and the superoxide dismutase activity in thoracic aorta. Hyperglycaemia and weight decrease were not affected. Genistein is able to reverse a diabetes established condition of allodynia, oxidative stress and inflammation, ameliorates NGF content and the vascular dysfunction, thus suggesting its possible therapeutic use for diabetes complications.

The purinergic antagonist PPADS reduces pain related behaviours and interleukin-1β, interleukin-6, iNOS and nNOS overproduction in central and peripheral nervous system after peripheral neuropathy in mice

&NA; Neuropathic pain consequent to peripheral injury is associated with local inflammation and overexpression of nitric oxide synthases (NOS) and inflammatory cytokines in locally recruited macrophages, Schwann and glial cells. We investigated the time course and localization of nitric oxide synthases (NOS) and cytokines in the central (spinal cord and thalamus) and peripheral nervous system (nerve and dorsal root ganglia), in a mouse model of mononeuropathy induced by sciatic nerve chronic constriction injury. ATP is recognized as an endogenous pain mediator. Therefore we also evaluated the role of purinergic signalling in pain hypersensitivity employing the P2 receptor antagonist, pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), on pain behaviour, NOS and cytokines. The PPADS daily administration starting on day 3 after injury dose‐ and time‐dependently decreased both tactile allodynia and thermal hyperalgesia. PPADS (25 mg/kg) completely reversed nociceptive hypersensitivity and simultaneously reduced the increased NO/NOS system and IL‐1β in both peripheral (injured sciatic nerve and L4–L6 ipsilateral dorsal root ganglia) and central steps of nervous system (L4–L6 spinal cord and thalamus) involved in pain signalling. IL‐6 was overexpressed only in the peripheral nervous system and PPADS prolonged administration reduced it in sciatic nerve. In conclusion, we hypothesize that NO/NOS and IL‐1β have a pronociceptive role in this neuropathy model, and that purinergic antagonism reduces pain hypersensitivity by inhibiting their overactivity.

Therapeutic effect of the endogenous fatty acid amide, palmitoylethanolamide, in rat acute inflammation: inhibition of nitric oxide and cyclo‐oxygenase systems

The anti‐inflammatory activity of the endogenous fatty acid amide palmitoylethanolamide and its relationship to cyclo‐oxygenase (COX) activity, nitric oxide (NO) and oxygen free radical production were investigated in the rat model of carrageenan‐induced acute paw inflammation and compared with the nonsteroidal anti‐inflammatory drug (NSAID) indomethacin. Palmitoylethanolamide (1, 3, 5, 10 mg kg−1; p.o.) and indomethacin (5 mg kg−1; p.o.) were administered daily after the onset of inflammation for three days and the paw oedema was measured daily; 24 h after the last dose (fourth day) the rats were killed and the COX activity and the content of nitrite/nitrate (NO2−/NO3−), malondialdehyde (MDA), endothelial and inducible nitric oxide synthase (eNOS and iNOS) were evaluated in the paw tissues. Palmitoylethanolamide had a curative effect on inflammation, inhibiting the carrageenan‐induced oedema in a dose‐ and time‐dependent manner. This effect was not reversed by the selective CB2 receptor antagonist (N‐[(1S)‐endo‐1,3,3‐trimethylbicyclo[2.2.1]heptan‐2yl]‐5‐(4‐chloro‐3‐methylphenyl)‐1‐(4‐methylbenzyl)pyrazole‐3 carboxamide) (SR144528), 3 mg kg−1 p.o. On the fourth day after carrageenan injection, COX activity and the level of NO2−/NO3−, eNOS and MDA were increased in the inflamed paw, but iNOS was not present. Palmitoylethanolamide (10 mg kg−1) and indomethacin markedly reduced these increases. Our findings show, for the first time, that palmitoylethanolamide has a curative effect in a model of acute inflammation. The inhibition of COX activity and of NO and free radical production at the site of inflammation might account for this activity.

Effect of the cannabinoid CB1 receptor antagonist, SR141716, on nociceptive response and nerve demyelination in rodents with chronic constriction injury of the sciatic nerve.

&NA; Many reports have shown the efficacy of cannabinoid agonists in chronic pain, whereas no report exists concerning the potential effect of cannabinoid antagonists following prolonged treatment. We tested the effects of repeated administration of the selective cannabinoid receptor type 1 (CB1) antagonist, SR141716 (rimonabant), in rats with chronic constriction injury of the sciatic nerve (CCI), an animal model of neuropathic pain. The repeated oral administration of SR141716 (1, 3 and 10 mg/kg, once a day for 1 week, from day 7 after the injury) dose dependently attenuated both thermal and mechanical hyperalgesia. A similar effect was observed in CCI wild‐type mice, whereas SR141716 was unable to elicit pain relief in CB1 knockout mice, suggesting CB1 receptors involvement in the SR141716‐induced antihyperalgesia. The antihyperalgesic activity of SR141716 was associated with a significant reduction of several pro‐inflammatory and pro‐nociceptive mediators such as tumor necrosis factor alpha (TNFα), prostaglandin‐E2 (PGE2), lipoperoxide and nitric oxide (NO) levels. The histological analysis of sciatic nerve sections showed a marked degeneration of myelinated fibers in CCI rats, which was substantially reduced after repeated administration of SR141716. This suggests that the compound may favour myelin repair and consequently promote long‐lasting functional recovery. This was confirmed by the maintenance of recovery for at least four weeks after treatment discontinuation. In conclusion, the present findings suggest that SR141716 is effective not only in alleviating neuropathic pain but also in favouring the nerve myelin repair.

The inhibition of monoacylglycerol lipase by URB602 showed an anti-inflammatory and anti-nociceptive effect in a murine model of acute inflammation.

2‐arachidonoylglycerol (2‐AG) is an endocannabinoid whose hydrolysis is predominantly catalysed by the enzyme monoacylglycerol lipase (MAGL). The development of MAGL inhibitors could offer an opportunity to investigate the anti‐inflammatory and anti‐nociceptive role of 2‐AG, which have not yet been elucidated. On these bases, URB602, a MAGL inhibitor, was tested in a murine model of inflammation/inflammatory pain.

Repeated treatment with the synthetic cannabinoid WIN 55,212-2 reduces both hyperalgesia and production of pronociceptive mediators in a rat model of neuropathic pain

The antinociceptive properties of cannabinoids in persistent pain are not fully elucidated. We investigated the effect of repeated treatment with the synthetic cannabinoid receptor agonist WIN 55,212‐2 on the neuropathic pain induced in rats by chronic constriction of the sciatic nerve. WIN 55,212‐2 administered daily throughout the development of neuropathy reversed the hyperalgesia, at a dose (0.1 mg kg−1, s.c.) that had no effect on the nociceptive responses of either paw contralateral to the sciatic ligation or of animals subjected to sham surgery. At 14 days after injury, the levels of mediators known to be involved in neuropathic pain, such as prostaglandin E2, NO and the neuronal NOS, were increased. Repeated treatment with WIN 55,212‐2 abolished these increases. In the light of the current clinical need for neuropathic pain treatments, these findings indicate that cannabinoid agonists, at doses devoid of psychoactive effects, could constitute important compounds for the development of new analgesics.

Genistein, a natural phytoestrogen from soy, relieves neuropathic pain following chronic constriction sciatic nerve injury in mice : anti-inflammatory and antioxidant activity

There is great interest in soy isoflavones as alternatives to endogenous estrogens not only in hormonal pathologies, but also in inflammatory, neurodegenerative diseases, and pain. We investigated the effect of the isoflavone genistein on neuropathic pain. Genistein binds estrogen receptors (ER) with higher affinity for the ERβ particularly expressed in neuronal and immune cells. Neuropathy was induced in mice by means of chronic sciatic nerve constriction, and the subcutaneous administration of genistein from the third day after the lesion reversed pain hypersensitivity in a time‐ and dose‐dependent manner. This effect may have been due to the activation of classical nuclear receptor and/or anti‐oxidant, anti‐inflammatory, and immunomodulating properties of genistein. The fact that a specific ERβ antagonist prevented both its anti‐allodynic and anti‐hyperalgesic action, whereas a specific ERα antagonist was ineffective and a non‐selective ER antagonist only reversed the anti‐allodynic effect, suggests the involvement of ERβ. Antioxidant effects are also involved as the anti‐nociceptive dose reversed the increase in reactive oxygen species and malondialdehyde in injured paw tissues, and increased the activity of anti‐oxidant enzymes. The phytoestrogen had immunomodulatory and anti‐inflammatory activities as it reduced peripheral and central nuclear factor‐κB, nitric oxide system and pro‐inflammatory cytokine over‐activation. Taken together, our results suggest that genistein could ameliorate painful neuropathy by multiple mechanisms.