Activation of CB2 receptors as a potential therapeutic target for migraine: evaluation in an animal model

Experimental evidence suggests an important role for endocannabinoids in pain modulation. Administration of endocannabinoids, either systemically or directed at appropriate pain relay or modulatory sites, alters pain sensitivity and changes the processing of nociceptive information within discrete spinal and brain pathways. Therefore, the discovery of endocannabinoid system has prompted the development of a range of novel cannabinoid receptor agonists and antagonists, some of which show marked selectivity for CB₁ or CB₂ receptors. CB₁ receptors are found in the central nervous system and have also been suggested to lie on peripheral nerve terminals [1]. Anandamide (AEA) is a full agonist at CB₁ receptors and a partial agonist at CB₂ receptors. Administration of AEA into the ipsilateral hindpaw of the rat reduced carrageenan-induced hyperalgesia [2] or formalin-induced nociception [3], indicating that activation of peripheral CB₁ receptors and in part CB₂ receptors, produces antinociception. CB₂ receptors are mostly located outside of the central nervous system [4, 5], and they were originally believed to be restricted to the periphery, primarily in the immune system, including mast cells, B and T cells, macrophages, and natural killer cells [6]. However, low levels of CB₂ receptors were also observed in different CNS regions, with an upregulation in sites implicated in nociception [7, 8]. Selective agonists for CB₂ receptor have been shown to produce analgesic effects in preclinical models of inflammatory, neuropathic, and bone cancer pain [9, 10]. Recent evidence shows that selective agonists of CB₂ receptors reduce nociception in a variety of preclinical models without producing tolerance [11, 12] or central side-effects [13], thus suggesting that CB₂ agonists may represent an attractive therapeutic target for pain.

A deficiency of the endocannabinoid system has been postulated to underlie the pathophysiology of migraine, as suggested by clinical studies, although biochemical studies providing a scientific basis for the potential efficacy of endocannabinoids in migraine are so far really limited [14]. Using a well characterized animal model of migraine [15‐18] based on the quantification of behavioral (nocifensive) and neurochemical changes induced by systemic nitroglycerin (NTG) - a vasodilator known to induce migraine-like headache in migraineurs [19‐21] - we have gathered evidence to suggest a derangement of the endocannabinoid function in migraine. In this frame, NTG-induced hyperalgesia is associated with an increased activity of the enzymes involved in the catabolism of endocannabinoids in several brain areas and with an increased density of CB binding sites in the mesencephalon [22]. Additionally, AEA has proved to be effective in preventing both NTG-induced activation (c-Fos) of neurons in the nucleus trigeminalis caudalis (NTC) and NTG-induced hyperalgesia at the formalin test [23]. This model has been tested over the years with different drugs and is generally accepted as a reliable animal model of migraine [15‐18, 24]. Several lines of evidence suggest the existence of a condition of trigeminal sensitization in migraineurs, which results in hyperalgesia, allodynia, and cognitive dysfunction during and between episodes [25, 26]. NTG is known to induce spontaneous-like headache attacks in migraine sufferers [16], probably as a consequence of sensitization phenomena [27]. In addition, NTG administration to migraineurs is associated to a significant facilitation in temporal summation of pain (reduced temporal summation threshold and increased painful sensation) when compared to baseline, to placebo condition and to controls. This finding suggests that migraineurs bear a susceptibility to develop migraine attack after NTG administration as a specific trait linked to a supersensitivity of the pain system to NTG [28]. AM1241 is a well characterized agonist of the CB₂ receptor that mediates antinociception following systemic administration in animal models not specific for migraine [29]. The present study is aimed at evaluating the potential analgesic effect of AM1241 in animal models of hyperalgesia induced by NTG administration.

Adult male Sprague-Dawley rats (weight 250-270 g, N = 4-8 for group) were evaluated in the present experiments. The principles of the Helsinki declaration and IASP’s guidelines for pain research in animals were rigorously applied [30]. The experimental research on animals was approved by ethics committee for research on animals of the University of Pavia (Document n. 2, 2012). Animals were housed in plastic boxes in groups of 3 with water and food available ad libitum and kept on a 12:12 hours light-dark cycle. All the rats were acclimatized to the test chamber before testing began.

Several lines of evidence have highlighted the importance of cannabinoid-mediated analgesia for nociceptive processing. Exogenous cannabinoids reduce responsiveness to noxious thermal, chemical and mechanical stimuli in rats. Cannabinoids induce antinociception by acting in neuroanatomical regions subserving transmission and modulation of pain signals, including, the periaqueductal gray (PAG) [33, 34] and the basolateral nucleus of amygdala [35]. The analgesic effect of endocannabinoids can be attributed in part to a neuronal mechanism based on the activation of CB₁ receptors expressed in primary afferent neurons. Indeed, it has been clearly demonstrated that CB₁ receptors are involved in modulation of pain signals via the inhibition of the release of neurotransmitters such as γ-aminobutyric acid (GABA), glutamate, dopamine, noradrenaline and acetylcholine [36]. Also CB₂ receptors appear to contribute to the analgesic effect, as suggested by the attenuation of pain in animal models of inflammatory and nociceptive pain [37]. Originally described in immune cells [4], CB₂ receptors have been demonstrated in human peripheral nerves after injury, as well as in brain regions, i.e. brainstem, which are particularly relevant for nociceptive integration [38‐41].

Endocannabinoid deficit may be involved in the pathophysiology of migraine as supported by an increasing body of evidence [22, 23]. In previous studies, using the same animal model proposed in the present study, we have provided data supporting the role of endocannabinoids in NTG-induced hyperalgesia [22]. In particular, we have demonstrated that NTG injection causes specific changes in endocannabinoids content in discrete cerebral areas, while the administration of AEA (20 mg kg^−1, i.p.), a CB₁/CB₂ agonist, reduces NTG-induced c-Fos expression in the NTC [22].

The analgesic effects derived from selective stimulation of CB₁ receptors is well known, but the relevant side-effects related to the stimulation of CB₁ receptors [11] have limited the clinical development of this therapeutic line. Interestingly, CB₂ agonists can reduce nociception in several preclinical models of pain without producing tolerance [9] or central side-effects [11]. Therefore CB2 antagonists seems more attractive as potential targets for modulating pain.

In the present study, we have shown that activation of CB₂ receptor, by means of AM1241 administration, induces analgesia at both the tail flick and the formalin tests. These findings are in agreement with behavioural, electrophysiological and neurochemical studies that showed the role for CB₂ receptor activation in modulating inflammatory nociception [40, 42‐44].

Furthermore, we detected an anti-hyperalgesic effect of AM1241 when the experimental paradigm was represented by phase II of formalin test in NTG treated animals, i.e. a condition of hyperalgesia mediated by the peripheral release of inflammation mediators and by central modulation.

The formalin test adopted in this study is based on the stimulation of sensorial areas that are outside of the trigeminal distribution, which may limit the applicability of our findings to migraine. However the model seems quite specific for cephalic pain since we have previously demonstrated that plantar injection of formalin in rats induces significant changes in calcitonin gene-related peptide (CGRP) immunoreactivity in the superficial laminae I and II ipsilateral to the injection side also in the NTC and that systemic NTG administration causes a reduction in CGRP-imunoreactivity in the NTC, but not in the lumbar dorsal horns [45]. Taken together with the demonstrated capability of NTG to induce spontaneous-like migraine attacks in migraineurs, these findings suggest that NTG-potentiated formalin test is a relevant model for investigating migraine circuitry. Though so far unexplained, the observation that a nociceptive stimulus delivered at the paw level is associated with NTC activation is further reported by the study of Han et al., [46], which showed that formalin injection in the paw induces Fos expression in NTC, and in other brainstem areas (i.e locus coeruleus) known to be involved in the modulation of migraine pain [47]. The possible mechanisms underlying NTG-induced hyperalgesia are presently elusive. However, it is becoming increasingly evident that NTG exerts its hyperalgesic effect through central and peripheral mechanisms [15, 16]. NTG may induce indeed a direct hyperalgesic effect via the formation of nitric oxide (NO) and via CGRP release in the NTC [42, 48], or indirectly via the activation of NOS synthesis at the meningeal level as a consequence of a sensitization of the trigeminovascular system [49‐51].

CB₂ receptors are expressed predominantly, but not exclusively, outside the CNS [41, 42], where they are localized extensively to cells of the immune system [52]. CB₂ receptors have been detected in cultured DRG neurons and in afferent fibers in the dorsal horn of the spinal cord [53], which confirms that CB₂ receptors are present in an area that is relevant for the mediation of the response to the formalin test.

In our experimental paradigm, CB₂ receptor activation may interfere with the mechanisms associated with NTG-induced hyperalgesia at two levels: central and peripheral. As regards the central one, Beltramo et al., [40] demonstrated that AM1241 reduces capsaicin-induced CGRP release in dorsal root ganglia (DRG), confirming that CB₂ activation may elicit cause analgesia by acting not only at non-neuronal peripheral sites but also at the neural level. Accordingly, the activation of CB₂ receptors reduces spinal fos protein expression and pain behaviour in a rat model of inflammation [42]. Taken together, these observations suggest that one possible mechanism through which AM1241 reduces NTG-induced hyperalgesia is the inhibition of CGRP release in laminae I and II of the NTC (lower brainstem and cervical spinal cord) [45], although further studies are needed to confirm this hypothesis. Indeed, following NTG administration, CGRP immunoreactivity decreased steadily in the NTC, whereas substance P immunoreactivity increased transiently [45].

When considering the potential peripheral mechanisms underlying the inhibitory effect of CB₂ agonism upon NTG-induced hyperalgesia, it seems noteworthy that emerging literature implicates a role for neuroimmune interactions in contributing to the development or maintenance of pathological pain states [54], and that it has been shown that NTG administration causes a delayed meningeal inflammation, as showed by activation of inducible NO synthases (iNOS) in macrophages of rodents, and a prolonged cold allodynia and heat hyperalgesia with a time-course consistent with NTG-induced migraine attacks [49, 55]. Therefore, we suggest that another possible site of action for AM1241 is represented by the dura, where the CB₂ agonist may counteract NO-induced activation of macrophages via the inhibition of NO by iNOS. Indeed, activation of CB2 receptors on non-neuronal cells has been postulated to suppress the release of inflammatory mediators that sensitize nociceptors [56]. This hypothesis is partially supported by the demonstrated interactions between the endocannabinoid and the nitrergic systems in CB₂ signalling. The activation of the CB₂ receptor seems indeed to be associated with a reduced expression of iNOS [57]. Additionally, CB₂ stimulation seems to suppress the release of pro-inflammatory factors such as NO and tumor necrosis factor alpha (TNF-α) in macrophages [58].

A collateral finding of this study is represented by the increase in nocifensive behavior in phase I of the formalin test following the administration of DMSO. This finding is partially in agreement with the observations of Colucci et al. (2008) [59] who showed an increase in the nocifensive behavior in both phases of the formalin test when DMSO was applied subcutaneously in the mouse paw. Conversely, the same study demonstrated an anti-nociceptive activity when DMSO was administered centrally or intraperitoneally [59]. These results suggest that DMSO displayed opposite effects on nociception and inflammation, depending on the route of administration. DMSO is one of the most common solvents used experimentally to dissolve hydrophobic substances for in vivo and in vitro purposes. The exact mechanism underlying DMSO pro-nociceptive activity is not known, but our findings show that the pro-nociceptive effect observed in phase I of the formalin test in the DMSO and NTG + DMSO groups (Figure 3) was counteracted by activation of CB2 receptors.

The present study lends further support to the therapeutic potential in migraine of probes that interfere with the endocannabinoid system. More specifically, stimulation of CB₂ receptors seems promising as it counteracts NTG-induced hyperalgesia in phase II of the formalin test and it is theoretically less likely to induce CNS side effects. However, the impact of long term treatment with CB2 agonists on their anti-hyperalgesic efficacy and on their effect on the immune system function remains to be elucidated.