Acetaldehyde via CGRP receptor and TRPA1 in Schwann cells mediates ethanol-evoked periorbital mechanical allodynia in mice: relevance for migraine

Abstract

Background: Ingestion of alcoholic beverages is a known trigger of migraine attacks. However, whether and how ethanol exerts its pro-migraine action remains poorly known. Ethanol stimulates the transient receptor potential vanilloid 1 (TRPV1) channel, and its dehydrogenized metabolite, acetaldehyde, is a known TRP ankyrin 1 (TRPA1) agonist.

Methods: Periorbital mechanical allodynia following systemic ethanol and acetaldehyde was investigated in mice after TRPA1 and TRPV1 pharmacological antagonism and global genetic deletion. Mice with selective silencing of the receptor activated modifying protein 1 (RAMP1), a component of the calcitonin gene-related peptide (CGRP) receptor, in Schwann cells or TRPA1 in dorsal root ganglion (DRG) neurons or Schwann cells, were used after systemic ethanol and acetaldehyde.

Results: We show in mice that intragastric ethanol administration evokes a sustained periorbital mechanical allodynia that is attenuated by systemic or local alcohol dehydrogenase inhibition, and TRPA1, but not TRPV1, global deletion, thus indicating the implication of acetaldehyde. Systemic (intraperitoneal) acetaldehyde administration also evokes periorbital mechanical allodynia. Importantly, periorbital mechanical allodynia by both ethanol and acetaldehyde is abrogated by pretreatment with the CGRP receptor antagonist, olcegepant, and a selective silencing of RAMP1 in Schwann cells. Periorbital mechanical allodynia by ethanol and acetaldehyde is also attenuated by cyclic AMP, protein kinase A, and nitric oxide inhibition and pretreatment with an antioxidant. Moreover, selective genetic silencing of TRPA1 in Schwann cells or DRG neurons attenuated periorbital mechanical allodynia by ethanol or acetaldehyde.

Conclusions: Results suggest that, in mice, periorbital mechanical allodynia, a response that mimics cutaneous allodynia reported during migraine attacks, is elicited by ethanol via the systemic production of acetaldehyde that, by releasing CGRP, engages the CGRP receptor in Schwann cells. The ensuing cascade of intracellular events results in a Schwann cell TRPA1-dependent oxidative stress generation that eventually targets neuronal TRPA1 to signal allodynia from the periorbital area.

Keywords: CGRP; Ethanol; Migraine; Oxidative stress; Schwann cell; TRPA1.

Fig. 1

Acetaldehyde sustains ethanol (EtOH)-evoked periorbital mechanical allodynia (PMA). a Dose-dependent motor coordination and b dose- and time-dependent PMA evoked by EtOH (intragastric, i.g.) or vehicle (veh, 0.9% NaCl) in C57BL/6J mice. c PMA evoked by EtOH (15%, 4 ml/kg, i.g.) or veh in C57BL/J mice pretreated (30 min before) with capsazepine (CPZ, 4 mg/kg, i.p.) or veh. d PMA evoked by EtOH (15%, 4 ml/kg, i.g.) or veh in Trpv1^+/+ and Trpv1^−/− mice. e, f PMA evoked by EtOH (15%, 4 ml/kg, i.g.) or veh in C57BL/6J mice pretreated (30 min before) with 4-methylpyrazole (4-M, 50 mg/kg, i.g. or 100 nmol, periorbital, p.orb.), or veh (n = 8 mice for behavioral experiments). g Acetaldehyde (ACD) levels in the infraorbital trunk of the trigeminal nerve of C57BL/6J mice receiving EtOH (15%, 4 ml/kg, i.g.) (n = 4 mice for each condition). h ACD levels in the infraorbital trunk of the trigeminal nerve of C57BL/6J mice 3 h after EtOH (15%, 4 ml/kg, i.g.) or veh and pretreated (30 min before) with 4-M (50 mg/kg, i.g. or 100 nmol, p.orb.) (n = 4 mice for each condition). Dash (-) is the combination of veh. *P < 0.05 vs. veh, time 0; ^#P < 0.05 vs. 4-Mp-EtOH, one and two-way ANOVA with Bonferroni post-hoc correction

Fig. 2

TRPA1 mediates periorbital mechanical allodynia (PMA) evoked by ethanol (EtOH) and acetaldehyde (ACD). a Dose- and time-dependent PMA evoked by ACD (i.p.) or vehicle (veh, 0.9% NaCl) in C57BL/6 J mice. b, c PMA evoked by ACD (0.1 mg/kg, i.p.), EtOH (15%, 4 ml/kg, i.g.), or veh in Trpa1^+/+ and Trpa1^−/− mice. d, e PMA evoked by ACD (0.1 mg/kg, i.p.), EtOH (15%, 4 ml/kg, i.g.), or veh in C57BL/6J mice treated (2 h after) with A967079 (A96 300 nmol, periorbital, p.orb.) or veh. (f, g) PMA evoked by ACD (0.1 mg/kg, i.p.), EtOH (15%, 4 ml/kg, i.g.), or veh in C57BL/6J mice treated (2 h after) with A96 (100 mg/kg, i.p.) or veh. (n = 8 mice). *P < 0.05 vs. veh; ^#P < 0.05 vs. Trpa1^+/+-EtOH, Trpa1^+/+-ACD, veh A96-EtOH, ACD, two-way ANOVA with Bonferroni post-hoc correction

Fig. 3

Schwann cell CGRP receptor activation mediates periorbital mechanical allodynia (PMA) evoked by ethanol (EtOH) and acetaldehyde (ACD). a, b Time-dependent PMA evoked by EtOH (15%, 4 ml/kg, i.g.), ACD (0.1 mg/kg, i.p.), or vehicle (veh, 0.9% NaCl) in C57BL/6J mice treated (30 min before) with olcegepant (1 nmol, periorbital, p.orb.) or veh. c, d PMA evoked by EtOH (15%, 4 ml/kg, i.g.), acetaldehyde (ACD, 0.1 mg/kg, i.p.), or veh in Plp1-Cre^ERT+;Ramp1^fl/fl and Plp1-Cre^ERT−;Ramp1^fl/fl (control) mice. PMA evoked by EtOH (15%, 4 ml/kg, i.g.), acetaldehyde (ACD, 0.1 mg/kg, i.p.), or vehicle (veh, 0.9% NaCl) in C57BL/6J mice treated (30 min before) with e, f SQ-22536 (25 nmol, p.orb.), g, h H89 (45 nmol, p.orb.), i, j L-NAME (1 mmol, p.orb.), k, l cPTIO (200 nmol, p.orb.), or veh. (n = 8 mice). *P < 0.05 vs. veh; ^#P < 0.05 vs. veh olcegepant, SQ-22536, H89, L-NAME, cPTIO, control -EtOH, two-way ANOVA with Bonferroni post-hoc correction

Fig. 4

Schwann cell TRPA1 receptor activation mediates periorbital mechanical allodynia (PMA) evoked by ethanol (EtOH) and acetaldehyde (ACD). a, b Time-dependent PMA evoked by EtOH (15%, 4 ml/kg, i.g.), ACD (0.1 mg/kg, i.p.), or vehicle (veh, 0.9% NaCl) in Plp1-Cre^ERT+;Trpa1^fl/fl and Plp1-Cre^ERT−; Trpa1^fl/fl (control) mice. c, d H₂O₂ levels in the infraorbital trunk of the trigeminal nerve of C57BL/6J mice receiving EtOH (15%, 4 ml/kg, i.g.), ACD (0.1 mg/kg, i.p.) or veh. e Representative photomicrographs and mean fluorescence intensity of 4-hydroxy-nonenal (4-HNE) staining in the infraorbital trunk of the trigeminal nerve of C57BL/6J mice receiving EtOH (15%, 4 ml/kg, i.g.), ACD (0.1 mg/kg, i.p.), or veh (0.9% NaCl) (scale bar: 10 µm) (n = 4 mice for each condition). PMA evoked by f, g EtOH (15%, 4 ml/kg, i.g.), h, i ACD (0.1 mg/kg, i.p.) or veh in C57BL/6J mice treated (2 h after) with PBN (100 mg/kg, i.p. or 670 nmol, periorbital, p.orb.) or veh. j, k PMA evoked by EtOH (15%, 4 ml/kg, i.g.), ACD (0.1 mg/kg, i.p.), or veh in Adv-Cre⁺;Trpa1^fl/fl and Adv-Cre⁻;Trpa1^fl/fl (control) mice. (n = 8 mice). *P < 0.05 vs. veh, time 0; ^#P < 0.05 vs. control -EtOH, ACD, Adv-Cre⁻;Trpa1^fl/fl -EtOH, ACD, veh PBN-EtOh, ACD

Fig. 5

Schematic representation of the pathway that signals cutaneous allodynia elicited by the ethanol (EtOH) metabolite, acetaldehyde (ACD). ACD, by targeting TRPA1, releases CGRP from trigeminal nerve terminals that activate CLR/RAMP1 on Schwann cells. CLR/RAMP1 activation elicits a transient cAMP-mediated protein kinase A (PKA) phosphorylation of endothelial nitric oxide synthase (eNOS). The ensuing nitric oxide (NO) release targets the oxidant-sensitive channel, TRPA1, in Schwann cells, which sustains and amplifies reactive oxygen species (ROS) generation. The persistent oxidative stress signals mechanical allodynia via TRPA1 on adjacent nerve fibers. ADH alcohol dehydrogenase