A pro-nociceptive phenotype unmasked in mice lacking fatty-acid amide hydrolase

Abstract

Fatty-acid amide hydrolase (FAAH) is the major enzyme responsible for degradation of anandamide, an endocannabinoid. Pharmacological inhibition or genetic deletion of FAAH (FAAH KO) produces antinociception in preclinical pain models that is largely attributed to anandamide-induced activation of cannabinoid receptors. However, FAAH metabolizes a wide range of structurally related, biologically active lipid signaling molecules whose functions remain largely unknown. Some of these endogenous lipids, including anandamide itself, may exert pro-nociceptive effects under certain conditions. In our study, FAAH KO mice exhibited a characteristic analgesic phenotype in the tail flick test and in both formalin and carrageenan models of inflammatory nociception. Nonetheless, intradermal injection of the transient receptor potential channel V1 (TRPV1) agonist capsaicin increased nocifensive behavior as well as mechanical and heat hypersensitivity in FAAH KO relative to wild-type mice. This pro-nociceptive phenotype was accompanied by increases in capsaicin-evoked Fos-like immunoreactive (FLI) cells in spinal dorsal horn regions implicated in nociceptive processing and was attenuated by CB1 (AM251) and TRPV1 (AMG9810) antagonists. When central sensitization was established, FAAH KO mice displayed elevated levels of anandamide, other fatty-acid amides, and endogenous TRPV1 agonists in both paw skin and lumbar spinal cord relative to wild-type mice. Capsaicin decreased spinal cord 2-AG levels and increased arachidonic acid and prostaglandin E2 levels in both spinal cord and paw skin irrespective of genotype. Our studies identify a previously unrecognized pro-nociceptive phenotype in FAAH KO mice that was unmasked by capsaicin challenge. The heightened nociceptive response was mediated by CB1 and TRPV1 receptors and accompanied by enhanced spinal neuronal activation. Moreover, genetic deletion of FAAH has a profound impact on the peripheral and central lipidome. Thus, genetic deletion of FAAH may predispose animals to increased sensitivity to certain types of pain. More work is necessary to determine whether such changes could explain the lack of efficacy of FAAH inhibitors in clinical trials.

Keywords: FAAH knockout; Fatty-acid amide hydrolase; TRPV1; anandamide; cannabinoid CB1 receptor; capsaicin; endocannabinoid; endovanilloid.

Figure 1.

Genetic deletion of FAAH produces an analgesic phenotype in response to hot water tail immersion, intraplantar carrageenan and intraplantar formalin. FAAH KO mice display longer withdrawal latencies in response to hot water tail immersion relative to WT mice (a). ^#p < 0.05 versus WT, 2-tailed t test. Five hours post i.pl. carrageenan, FAAH KO mice show reduced thermal hyperalgesia in the paw ipsilateral, but not contralateral, to carrageenan injection relative to WT mice (b). Data are expressed as ± SEM (n = 6 per group). ***p < 0.001 versus WT ipsilateral, 2-way ANOVA, Bonferroni post-hoc. I.pl. formalin produces increased composite pain scores in WT animals relative to FAAH KO animals (c). FAAH KO mice displayed decreased levels of pain behavior from 10 to 30 min post-injection relative to WT mice. *p < 0.05, **p < 0.01, ***p < 0.001 versus FAAH KO, 2-tailed t test. FAAH KO mice displayed decreases in the area under the curve in phase 2 of formalin-evoked pain behavior but no change during phase 1 (d). ***p < 0.001, 2-way ANOVA, Bonferroni post-hoc. FAAH KO: FAAH knockout; WT: wildtype; CPS: composite pain score; AUC: area under curve.

Figure 2.

FAAH KO mice show increases in capsaicin-evoked nocifensive behavior compared to WT littermates. FAAH KO mice display increased levels of nocifensive behavior relative to WT mice (a) in response to intraplantar administration of capsaicin (1 µg i.pl.). Responding did not differ between FAAH KO and WT animals that received local injections of vehicle (10 µl i.pl.) (b). Data are expressed as mean ± SEM (n = 6 per group). **p < 0.01 KO male and KO female versus WT male and WT female, One-way ANOVA followed by Newman–Keuls post-hoc test. FAAH KO: FAAH knockout; i.pl: intraplanar; WT: wildtype.

Figure 3.

FAAH KO mice show increased capsaicin-evoked heat hyperalgesia compared to WT littermates. FAAH KO mice exhibit a delayed resolution of capsaicin-induced heat hyperalgesia relative to WT mice (a). Heat hyperalgesia does not develop in the noninjected (contralateral) paw in either genotype (b). Paw withdrawal latencies produced by i.pl. vehicle administration do not differ in FAAH KO mice in the paw ipsilateral to (c) or contralateral to vehicle injection (d). Data are expressed as mean ± SEM (n = 6 per group). + p < 0.05 KO male versus WT female, one-way ANOVA followed by Newman–Keuls post hoc test. *p < 0.05 KO male and KO female versus WT male and WT female, ^$p < 0.05 WT female versus KO male and KO female, ^##p < 0.01 WT male versus KO male and KO female one-way ANOVA followed by Newman–Keuls post hoc test. @p < 0.05 KO female versus WT male and WT female, one-way ANOVA followed by Newman–Keuls post-hoc test. FAAH KO: FAAH knockout; i.pl: intrplanar; WT: wildtype.

Figure 4.

FAAH KO mice show enhanced capsaicin-evoked mechanical hypersensitivity relative to WT littermates. FAAH KO mice display, relative to WT mice, a prolongation of mechanical hypersensitivity in the paw ipsilateral (capsaicin-injected) (a), but not contralateral (b), to local injection of capsaicin (1 µg i.pl.). FAAH KO mice displayed an enhanced mechanical hypersensitivity relative to WT littermates at 30, 60, and 120 minutes post-capsaicin administration. I.pl. injection of vehicle did not alter mechanical paw withdrawal thresholds in the paw ipsilateral (c) or contralateral (d) to injection in either genotype. Data are expressed as mean ± SEM (n = 5–6 per group). *p < 0.05, **p < 0.01 FAAH KO versus WT, Two-tailed t test. FAAH KO: FAAH knockout; i.pl: intraplanar; WT: wildtype.

Figure 5.

FAAH KO mice display increases in capsaicin-evoked Fos-like immunoreactive cells in the spinal dorsal horn. Compared to WT mice, FAAH KO mice exhibit increased numbers of Fos-like immunoreactive cells in the superficial dorsal horn (lamina I and II), the nucleus proprius (lamina III and IV), and the neck region of the dorsal horn (lamina V-VI) but not in the ventral horn (a). Representative photomicrographs of capsaicin treated (i.pl.) WT (b) and FAAH KO mice (c). Data are expressed as mean ± SEM (n = 4 per group). ***p < 0.001, *p < 0.05 versus WT, 2-way ANOVA, Bonferroni post-hoc. FAAH KO: FAAH knockout; WT: wildtype.

Figure 6.

AM251 attenuates capsaicin-evoked nocifensive behavior in FAAH KO but not WT mice whereas AMG9810 increases it in WT but not FAAH KO mice. FAAH KO mice receiving vehicle (i.p.) show increased levels of capsaicin-evoked nocifensive behavior relative to FAAH KO mice receiving the CB1 antagonist AM251 (3 mg/kg i.p.) (a). AM251 (3 mg/kg, i.p.) pretreatment eliminated the increase in capsaicin-evoked nocifensive behavior in FAAH KO mice at a dose that did not reliably alter responding in WT mice (a). Data are expressed as mean ± SEM (n = 5–6 per group). Capsaicin-treated WT mice receiving vehicle (i.p.) display lower levels of nocifensive behavior compared to all other groups tested (b). Capsaicin-treated WT mice receiving TRPV1 agonist AMG9810 (3 mg/kg, i.p.) display lower levels of nocifensive behavior than capsaicin-treated FAAH KO mice receiving vehicle (i.p.) (b). Data are expressed as mean ± SEM (n = 5–6 per group). ^##p < 0.01 versus FAAH KO vehicle. Planned comparison, 2-tailed t test. ***p < 0.001, **p < 0.01, *p < 0.05 versus wt vehicle, ^#p < 0.05 versus FAAH KO vehicle. ANOVA, Newman–Keuls post-hoc. FAAH KO: FAAH knockout; WT: wildtype.

Figure 7.

FAAH KO mice receiving i.p. vehicle display a delayed resolution of capsaicin-evoked heat and mechanical hypersensitivity. FAAH KO mice receiving vehicle (i.p.) display a prolongation of capsaicinevoked heat hypersensitivity, as measured by thermal paw withdrawal latencies, relative to WT mice receiving vehicle (i.p.) (a). FAAH KO mice receiving vehicle (i.p.) display a prolongation of capsaicin-evoked mechanical hypersensitivity relative to WT animals similarly receiving vehicle (i.p.) (b). Data are expressed as mean ± SEM (n = 5–6 per group).***p < 0.001, **p < 0.01, *p < 0.05 versus FAAH KO vehicle, Two-tailed t test. FAAH KO: FAAH knockout; WT: wildtype.

Figure 8.

AM251 eliminates the pro-nociceptive phenotype of FAAH KO mice as measured by capsaicinevoked heat and mechanical hypersensitivity. The CB1 antagonist AM251 (3 mg/kg, i.p.) attenuates the heightened capsaicin-evoked heat hypersensitivity observed in FAAH KO mice compared to FAAH KO mice pretreated with vehicle (i.p.) (a). AM251 (3 mg/kg i.p.) does not alter capsaicin-evoked heat hyperalgesia in WT mice relative to WT mice receiving vehicle (i.p) (b). AM251 (3 mg/kg, i.p.) restores paw withdrawal latencies of FAAH KO mice to levels observed in WT mice (c). In FAAH KO mice, AM251 (3 mg/kg i.p.) reduced capsaicin-evoked mechanical hypersensitivity relative to vehicle treatment at 30, 60, and 120 minutes post-capsaicin administration (d). In WT mice, AM251 (3 mg/kg, i.p.) does not alter mechanical hypersensitivity relative to vehicle (i.p.) treatment (e). In FAAH KO mice, AM251 (3 mg/kg i.p.) restores paw withdrawal thresholds to levels observed in WT mice (f). Data are expressed as mean ± SEM (n = 5–6 per group). ***p < 0.001, **p < 0.01, *p < 0.05 versus FAAH KO vehicle, two-tailed t test. FAAH KO: FAAH knockout; WT: wildtype.

Figure 9.

AMG9810 eliminates the pro-nociceptive phenotype of FAAH KO mice as measured by capsaicin-evoked heat and mechanical hypersensitivity. In FAAH KO mice, the TRPV1 antagonist AMG9810 (3 mg/kg, i.p.) pretreatment promotes the resolution of capsaicin-evoked heat hypersensitivity relative to vehicle (i.p) treatment (A). In WT mice, AMG9810 (3 mg/kg, i.p.) does not alter capsaicin-evoked heat hypersensitivity relative to vehicle (i.p.) treatment (B). AMG9810 pretreatment in FAAH KO mice restores thermal paw withdrawal latencies to levels observed in vehicle-treated WT mice (C). In FAAH KO mice, AMG9810 (3 mg/kg i.p.) reduced capsaicin-evoked mechanical hypersensitivity relative to vehicle (i.p.) treatment (D). In WT mice, AMG9810 (3 mg/kg, i.p.) does not alter capsaicin-evoked mechanical hypersensitivity relative to vehicle (i.p.) treatment (E). FAAH KO mice receiving AMG9810 (3 mg/kg, i.p.) exhibit a lower magnitude of mechanical hypersensitivity at 5 min but enhanced mechanical hypersensitivity at 120 minutes post capsaicin relative to WT mice receiving vehicle (i.p) (F). Data are expressed as mean ± SEM (n = 5-6 per group). ***p < 0.001, **p < 0.01, *p < 0.05 vs. FAAH KO vehicle, ++p < 0.01 vs. WT vehicle, #p < 0.05 vs. FAAH KO AMG9810, Two-tailed t-test. FAAH KO: FAAH knockout; WT: wildtype.

Figure 10.

Capsaicin decreases, whereas genetic deletion of FAAH increases, N-acyl ethanolamines in paw skin and lumbar spinal cord. Paw skin levels of AEA, LEA, PEA, and SEA were elevated in FAAH KO relative to WT mice irrespective of capsaicin or vehicle (i.pl.) treatment (a, c, e, g). Paw skin SEA levels are preferentially lowered by capsaicin in FAAH KO but not WT mice. Whereas FAAH KO mice in general display higher levels of SEA than WT mice, capsaicin lowered paw skin SEA levels in FAAH KO but not WT mice (g). FAAH KO mice displayed elevated spinal cord levels of AEA, LEA, PEA and SEA relative to WT mice irrespective of capsaicin or vehicle (i.pl.) treatment (b, d, f, h). Data are expressed as mean + SEM (n = 6). Tissue was dissected 1 h following intradermal injection of vehicle or capsaicin. ^###p < 0.0001, ^##p < 0.01, ^#p < 0.05 versus WT. Two-way ANOVA.⁺⁺p < 0.01, ⁺p < 0.05 versus FAAH KO vehicle (K), one-way ANOVA followed by Newman–Keuls post hoc. FAAH KO: FAAH knockout; WT: wildtype.

Figure 11.

Capsaicin alters levels of arachidonic acid derivatives in both paw skin and lumbar spinal cord. Capsaicin increases paw skin levels of arachidonic acid (AA) and prostaglandin E2 (PGE2) in FAAH KO and WT mice (c, e). Capsaicin selectively increases paw skin PGF2α levels in FAAH KO but not WT mice (g). Capsaicin decreased 2-AG levels in lumbar spinal cord (b) but not paw skin (a). Capsaicin increased levels of AA and PGE2 in both paw skin (c, e) and lumbar spinal cord (d, f). Data are expressed as mean + SEM (n = 6). Tissue was dissected 1 h following intradermal injection of vehicle or capsaicin. ***p < 0.0001, *p < 0.05 versus vehicle. Two-way ANOVA.⁺p < 0.05 versus FAAH KO vehicle, one-way ANOVA followed by Newman–Keuls post hoc. FAAH KO: FAAH knockout; WT: wildtype.

Figure 12.

Genetic deletion of FAAH increases endovanilloids in lumbar spinal cord. Capsaicin increases paw skin DEA levels in both FAAH KO and WT mice (a). FAAH KO mice display higher spinal cord levels of DEA and OEA relative to WT mice irrespective of capsaicin or vehicle (i.pl.) treatment (b, d). Data are expressed as mean + SEM (n = 6). Tissue was dissected 1 h following intradermal injection of vehicle or capsaicin. *p < 0.05 versus vehicle, ^###p < 0.0001 versus WT. Two-way ANOVA; FAAH KO: FAAH knockout; WT: wildtype.

Figure 13.

Genetic deletion of FAAH alters levels of several orphan cannabinoid receptor ligands and endovanilloids detectable in lumbar spinal cord but not paw skin. In FAAH KO mice, lumbar spinal cord levels of N-arachidonoyl serine are increased (a) whereas N-arachidonoyl glycine levels are decreased relative to WT mice (b). Capsaicin differentially affects levels of the endovanilloid N-arachidonoyl taurine in FAAH KO mice. FAAH KO mice receiving capsaicin (i.pl.) display lower levels of N-arachidonoyl taurine compared to FAAH KO mice receiving vehicle. Capsaicin decreased spinal N-arachidonoyl taurine levels in FAAH KO mice but not WT mice. Levels of N-arachidonoyl taurine were higher in FAAH KO mice receiving vehicle than all other groups tested (c). FAAH KO mice display increased spinal cord levels of the endovanilloids N-docosahexaenoyl GABA (d) and N-linoleoyl GABA (e), and decreased levels of the endovanilloid Narachidonoyl GABA (f) relative to WT mice. Data are expressed as mean + SEM (n = 6). ^###p < 0.0001, ^##p < 0.01 versus WT. Two-way ANOVA. ⁺⁺⁺p < 0.0001 versus all other groups, ⁺⁺p < 0.01 versus WT capsaicin and WT vehicle, one-way ANOVA followed by Newman–Keuls post hoc. FAAH KO: FAAH knockout; WT: wildtype.