Epoxy fatty acids mediate analgesia in murine diabetic neuropathy

Abstract

Background: Neuropathic pain is a debilitating condition with no adequate therapy. The health benefits of omega-3 fatty acids are established, however, the role of docosahexaenoic acid (DHA) in limiting pain has only recently been described and the mechanisms of this action remain unknown. DHA is metabolized into epoxydocosapentanoic acids (EDPs) via cytochrome P450 (CYP450) enzymes which are substrates for the soluble epoxide hydrolase (sEH) enzyme. Here, we tested several hypotheses; first, that the antinociceptive action of DHA is mediated by the EDPs. Second, based on evidence that DHA and CYP450 metabolites elicit analgesia through opioid signalling, we investigated this as a possible mechanism of action. Third, we tested whether the analgesia mediated by epoxy fatty acids had similar rewarding effects as opioid analgesics.

Methods: We tested diabetic neuropathic wild-type and sEH null mice in a conditioned place preference assay for their response to EDPs, sEHI and antagonism of these treatments with naloxone, a mu-opioid receptor antagonist.

Results: The EDPs and sEH inhibitors were efficacious against chronic pain, and naloxone antagonized the action of both EDPs and sEH inhibitors. Despite this antagonism, the sEH inhibitors lacked reward side effects differing from opioids.

Conclusions: The EpFA are analgesic against chronic pain differing from opioids which have limited efficacy in chronic conditions.

Significance: EDPs and sEHI mediate analgesia in modelled chronic pain and this analgesia is blocked by naloxone. However, unlike opioids, sEHI are highly effective in neuropathic pain models and importantly lack rewarding side effects.

Figure 1

EDPs, the omega-3 DHA derived epoxidized metabolites, mediate analgesia in a model of diabetic neuropathic pain. Exogenous EDPs 1 mg/kg combined with an ineffective low dose of t-TUCB 1 mg/kg to block their rapid degradation by the sEH enzyme were significantly effective against neuropathic pain (t-TUCB 1 mg/kg alone appears in supplemental material). In the CPP assay this is indicated by increased time (seconds, y axis) spent in the drug paired chamber (One Way ANOVA, p= 0.041). Prior to the CPP assay diabetic mice were assessed for phenotypic allodynia indicating diabetic neuropathy in the von Frey assay. The results depicted below the graph are the grams of force to elicit a hind paw withdrawal (von Frey (gr)) of pre-streptozocin baseline scores (Naïve) and post streptozocin painful baseline scores (Diabetic) prior to the start of treatment in the CPP assay.

Figure 2

Analgesia mediated by EDPs and sEHI is blocked mu-opioid antagonism. The analgesia mediated by 1 mg/kg EDPs co-administered with low dose 1mg/kg t-TUCB was blocked by naloxone (1 mg/kg) a mu-opioid receptor antagonist. The sEH inhibitor t-TUCB blocks the degradation of multiple classes of EpFA in vivo and a single administration of 10 mg/kg dose effectively induced a CPP response indicating pain relief (One Way ANOVA, p≤0.001). In the CPP assay this is indicated by increased time (seconds, y axis) spent in the drug paired chamber. The efficacy of 10 mg/kg t-TUCB was also blocked by naloxone. There was no significant effect of the naloxone in control neuropathic mice. The diabetic mice were assessed for phenotypic allodynia indicating diabetic neuropathy in the von Frey assay prior to the CPP assay. The results depicted below the graph are the grams of force to elicit a hind paw withdrawal (von Frey (gr)) of pre-streptozocin baseline scores (Naïve) and post streptozocin painful baseline scores (Diabetic) prior to the start of treatment in the CPP assay.

Figure 3

Inhibitors of sEH do not significantly induce a CPP response in diabetic sEH null mice. In sEH null mice induced with diabetic neuropathy neither t-TUCB nor TPPU, a structurally distinct but similarly potent sEHI, have a significant effect in the CPP assay (One Way ANOVA, p=0.333). In the CPP assay this is measured by increased time (seconds, y axis) spent in the drug paired chamber. This is in contrast to the robust effect of t-TUCB in wild type diabetic mice (Fig 2) indicating that targeting the sEH enzyme mediates the sEHI efficacy. The diabetic mice were assessed for phenotypic allodynia indicating diabetic neuropathy in the von Frey assay prior to the CPP assay. The results depicted below the graph are the grams of force to elicit a hind paw withdrawal (von Frey (gr)) of pre-streptozocin baseline scores (Naïve) and post streptozocin painful baseline scores (Diabetic) prior to the start of treatment in the CPP assay.

Figure 4

The sEHI effectively mediates analgesia in neuropathic pain and outperforms celecoxib. (a) When the sEHI TPPU at 10mg/kg was tested in naïve mice it had no significant effect in the CPP assay. The same 10mg/kg dose of celecoxib seemed to induce a response but it was not significant (One Way ANOVA, p=0.132). In the CPP assay this is indicated by increased time (seconds, y axis) spent in the drug paired chamber. (b) When assessed in diabetic neuropathic mice the 10mg/kg TPPU showed a significant and robust response and celecoxib was without effect (One Way ANOVA, p=0.003). The diabetic mice were assessed for phenotypic allodynia indicating diabetic neuropathy in the von Frey assay. The results depicted below the graph (where applicable) are the grams of force to elicit a hind paw withdrawal (von Frey (gr)) of pre-streptozocin baseline scores (Naïve) and post streptozocin painful baseline scores (Diabetic) prior to the start of treatment in the CPP assay.