Anna Elisa Trovato

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 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.

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.

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.

AM404, an inhibitor of anandamide uptake, prevents pain behaviour and modulates cytokine and apoptotic pathways in a rat model of neuropathic pain

1 An attractive alternative to the use of direct agonists at the cannabinoid receptor type 1 (CB1) in the control of neuropathic pain may be to potentiate the actions of endogenous cannabinoids. Thus, the effects of AM404, an inhibitor of anandamide uptake, were assessed in an experimental model of neuropathic pain in rats. 2 Daily treatment with AM404 prevented, time‐ and dose‐dependently, the development of thermal hyperalgesia and mechanical allodynia in neuropathic rats. Antagonists at cannabinoid CB1 or CB2 receptors, or at the transient receptor potential vanilloid type 1 receptor, each partially reversed effects induced by AM404. A complete reversal was obtained when the three antagonists were given together, suggesting that all three receptors are involved. 3 AM404 treatment affected two pathways involved in the generation and maintenance of neuropathic pain, one mediated by nitric oxide (NO) and the other by cytokines. AM404 completely prevented the overproduction of NO and the overexpression of nNOS, inhibited the increase in tumour necrosis factor α (TNFα) and enhanced the production of interleukin‐10. Both NO and TNFα are known to contribute to the apoptotic process, which plays an important role in the establishment of chronic pain states. AM404 treatment prevented the increase in the ratio between pro‐ and anti‐apoptotic gene bax/bcl‐2 expression observed in the spinal cord of neuropathic rats. 4 Taken together, these findings suggest that inhibition of endocannabinoid uptake, by blocking the putative anandamide carrier, results in the relief of neuropathic pain and may represent a novel strategy for treating chronic pain.

Transient early expression of TNF-α in sciatic nerve and dorsal root ganglia in a mouse model of painful peripheral neuropathy

The proinflammatory cytokine tumor necrosis factor-alpha (TNF) is an important mediator in neuropathic pain. We investigated the temporal pattern of TNF mRNA expression in the sciatic nerve, in dorsal root ganglia (DRG) and spinal cord in the mouse chronic constriction injury model of neuropathy with quantitative real-time polymerase chain reaction. Neuropathic pain-like behaviour was monitored by evaluating thermal hyperalgesia and mechanical allodynia. Pain-related behaviour and TNF expression were evaluated 6 h, 1, 3, 7 and 14 days after injury. Naive animals and sham-operated mice were used as controls. We found an early upregulation of sciatic nerve TNF mRNA levels in chronic constriction injury (CCI) and sham-operated animals 6 h after surgery: 1 day later TNF overexpression was present in CCI mice only and disappeared 3 days after injury. The mRNA cytokine levels were elevated in DRG 1 and 3 days after surgery in CCI animals only, while the cytokine was not modulated in the spinal cord. A significant hyperalgesia was present in CCI and sham-operated mice at 6 h and 1 day, while at later time point only CCI mice presented lower thresholds. Mechanical allodynia was already present only in CCI animals 6 h from surgery and remained constant up to the 14 th day. The results indicate that a transient early TNF upregulation takes place in peripheral nervous system after CCI that can activate a cascade of proinflammatory/pronociceptive mediators.

Altered peripheral myelination in mice lacking GABAB receptors

Emerging evidence implicates gamma-aminobutyric acid type B (GABA(B)) receptors in peripheral nervous system (PNS) functions. In order to elucidate which biochemical, morphological and functional parameters of peripheral nerve fibers depend on GABA(B) receptors we studied GABA(B1)-deficient mice, which are devoid of functional GABA(B) receptors. Here, we show that GABA(B1)-deficient mice exhibit morphological and molecular changes in peripheral myelin, including an increase in the number of irregular fibers and increases in the expression levels of the myelin proteins PMP22 and P0. Moreover, the number of small myelinated fibers and small neurons of the lumbar dorsal root ganglia is higher in GABA(B1)-deficient mice than in wild-type littermates. We further show that GABA(B1)-deficient mice exhibit gait alterations and reduced allodynia. In summary, our findings implicate GABA(B) receptors in the PNS myelination process and raise the possibility that PNS alterations contribute to the sensory phenotypes of GABA(B1)-deficient mice.

Purinergic receptor antagonist modulates hyperalgesia and allodynia in a mouse model of neuropathy: Involvement of NO system and cytokine network at central and peripheral nervous system level

In response to infection, several species of reptiles and amphibians spend more time in basking, leading to an elevated body temperature, a thermoregulatory response known as behavioral or emotional fever (Kluger et al., 1975). A similar response can be elicited by injection of purified lipopolysaccharide (LPS) or even by the stress of handling in some cases (Deen and Hutchinson, 2000, Bicego et al., 2002, Cabanac and Gosselin, 1993). In mammals, a febrile response to infection is mediated, at least in part, by the secretion of IL-1 (Hansen et al., 2001). IL-1 acts in part on the hypothalamus in the brain, leading to a change in the body’s thermostat, elevating body temperature through increased metabolic rate (Hill et al., 2004). Such a response is also seen to LPS injection in mammals. Given that ectotherms exhibit the same end effect of infection or LPS injection, it may be that IL-1 is mediating behavioral fever, but this has not yet been investigated. Our research is investigating the possibility that IL-1 also elicits a febrile response in ectotherms by monitoring the body temperature and behavior of juvenile green iguanas (Iguana iguana) following systemic injection of IL-1.