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  • br Experimental procedures br Results br Discussion This stu

    2021-09-18


    Experimental procedures
    Results
    Discussion This study demonstrates that the genetic disruption or pharmacological inhibition of FAAH completely prevented all manifestations of pain symptoms in an NTG-induced migraine animal model. Thus, FAAH disruption or blockade abolished the activation of trigeminal neurons after NTG injection, as well as NTG-induced mechanical hyperalgesia. These results are in line with the previous demonstration that FAAH activity is increased, while AEA content was diminished in human migraine patients (Cupini et al., 2008, Rossi et al., 2008). FAAH is therefore an interesting pharmacological target for the treatment and prevention of migraine headaches. To examine if endocannabinoid signaling affects NTG-induced migraine-like pain, we first compared the effect of NTG in different knockout animal models of endocannabinoid system components. The Exendin-3 (9-39) amide of CB1, CB2 or MAGL did not modify the effect of NTG, whereas FAAH knockout showed a strikingly reduced hyperalgesia in the NTG model. As FAAH contributes to the degradation of AEA while MAGL primarily degrades 2-AG (Dinh et al., 2002, Makara et al., 2005), our results indicate that migraine is modulated by the endogenous AEA tone, but not dependent on 2-AG levels. Interestingly knockout of either CB1 or CB2 receptor did not modify NTG effect, however this may be due to the NTG dose, which already induced the maximum hyperalgesic effect in WT animals. We can envision two possible mechanisms to account for the effects of Exendin-3 (9-39) amide FAAH inhibition that are not mutually exclusive. First, metabolites generated by FAAH could be important for the induction of migraine-like symptoms in the NTG model. However, we believe that this is not likely to be the case, because exogenously administered AEA rescued the acute hyperalgesia in same animal model of migraine pain (Greco et al., 2010). Furthermore, at least in pulmonary arteries, FAAH-dependent AEA metabolites produce a vasoconstrictive response (Wenzel et al., 2013), which would counteract the vasodilatory response to NTG. Rather, we feel that the effects are best explained by an increased CB1 tone through elevated AEA levels. Indeed, the effects of FAAH inhibition were dependent on the activity of CB1 receptors and not present after pharmacological blockade of CB1. Thus, rimonabant restored the reduced NTG-induced hyperalgesia in both FAAH inhibitor-treated WT animals and FAAH knockout mice, as well as the trigeminal neuronal hyperactivity. Along with past studies showing inhibitory effect of CB1 activation on peripheral pain (Pertwee, 2001) and brain neuronal hyperactivity (Di Marzo et al., 1998), these results suggest that inhibition of vasodilator-induced pain and neuronal hyperactivity evoked by FAAH down-regulation was due to activation of CB1 receptor. A CB1-mediated mechanism is also in line with the long history of cannabis use for migraine treatment (Russo, 1998). Although analgesic effect of endogenous or exogenous CB1 receptor agonists are well studied (Pertwee, 2001), few recent and previous studies showed that activation of CB1 receptor can inhibit specific migraine-related symptoms. Activation of CB1 receptor but not CB2 receptor suppressed the KCl-induced cortical spreading depression in rat brain (Kazemi et al., 2012). Furthermore, brain CB1 receptor activation suppressed basal and dural-evoked Aδ-fiber neuron activity in the rat trigeminocervical complex, which was inhibited by antagonizing brain CB1 receptor or 5-HT1B/1D receptors, suggesting that therapeutic action of triptans may be mediated by endocannabinoid signaling (Akerman et al., 2013). We used two different FAAH inhibitors, URB597 and PF3845, and both showed similar dose-dependent analgesic potency despite of their different structures. These compounds were previously shown to produce antinociceptive effects in inflammatory and neuropathic pain models. Thus, systemically administered URB597 inhibits inflammatory pain at a low dose (0.3mg/kg) and neuropathic pain at a high dose (10mg/kg) (Jayamanne et al., 2006, Russo et al., 2007). PF3845 also showed analgesic effects in inflammatory pain models after systemic (i.p.) and topical (intraplantar) injection (Booker et al., 2012, Kinsey et al., 2010). Both compounds showed their maximal analgesic effects 2–6h after the injection, which co-incided with a 20–30-fold increase in brain and spinal cord AEA levels (Ahn et al., 2009b, Russo et al., 2007). Mice lacking FAAH also displayed a 15-fold increase of AEA levels and reduced pain sensitivity in the tail flick, hot plate and formalin tests (Cravatt et al., 2001), although we found no difference in mechanical pain thresholds. Together, these results demonstrate unequivocally that the inhibition of NTG-induced hyperalgesia is a direct consequence of FAAH inhibition and not due to compensatory effects in FAAH knockouts, or to off-target effects of the pharmacological compounds.