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. 2019 Mar 6;20(1):25.
doi: 10.1186/s10194-019-0978-z.

ROS/TRPA1/CGRP Signaling Mediates Cortical Spreading Depression

Affiliations
Free PMC article

ROS/TRPA1/CGRP Signaling Mediates Cortical Spreading Depression

Liwen Jiang et al. J Headache Pain. .
Free PMC article

Abstract

Objectives: The transient receptor potential ankyrin A 1 (TRPA1) channel and calcitonin gene-related peptide (CGRP) are targets for migraine prophylaxis. This study aimed to understand their mechanisms in migraine by investigating the role of TRPA1 in cortical spreading depression (CSD) in vivo and exploring how reactive oxygen species (ROS)/TRPA1/CGRP interplay in regulating cortical susceptibility to CSD.

Methods: Immunohistochemistry was used for detecting TRPA1 expression. CSD was induced by K+ on the cerebral cortex, monitored using electrophysiology in rats, and intrinsic optical imaging in mouse brain slices, respectively. Drugs were perfused into contralateral ventricle of rats. Lipid peroxidation (malondialdehyde, MDA) analysis was used for indicating ROS level.

Results: TRPA1 was expressed in cortical neurons and astrocytes of rats and mice. TRPA1 deactivation by an anti-TRPA1 antibody reduced cortical susceptibility to CSD in rats and decreased ipsilateral MDA level induced by CSD. In mouse brain slices, H2O2 facilitated submaximal CSD induction, which disappeared by the antioxidant, tempol and the TRPA1 antagonist, A-967079; Consistently, TRPA1 activation reversed prolonged CSD latency and reduced magnitude by the antioxidant. Further, blockade of CGRP prolonged CSD latency, which was reversed by H2O2 and the TRPA1 agonist, allyl-isothiocyanate, respectively.

Conclusions: ROS/TRPA1/CGRP signaling plays a critical role in regulating cortical susceptibility to CSD. Inhibition ROS and deactivation of TRPA1 channels may have therapeutic benefits in preventing stress-triggered migraine via CGRP.

Keywords: Calcitonin gene-related peptide; Cortical spreading depression; Migraine; Reactive oxygen species; Transient receptor potential ankyrin a 1.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Representative images showing immunohistochemistry detection of TRPA1 expression in neurons and astrocytes of rat cerebral cortex. Double immune-labelling showed TRPA1 expressed in cerebral cortical neurons (upper panel, white arrows) and astrocytes (lower panel, white arrows). DAPI staining indicates nucleus is shown in blue; TRPA1 in red, NeuN indicates neurons or GFAP indicates glial cells shown in green
Fig. 2
Fig. 2
Representative images showing immunohistochemistry detection of TRPA1 expression in neurons and astrocytes of mouse cerebral cortex. Double immune-labelling showed TRPA1 expressed in cerebral cortical neurons (upper panel, white arrows) and astrocytes (lower panel, white arrows). DAPI staining indicates nucleus is shown in blue; TRPA1 in red, NeuN indicates neurons or GFAP indicates glial cells shown in green
Fig. 3
Fig. 3
Effects of the anti-TRPA1 antibody, perfused into the contralateral ventricle, on cortical susceptibility to CSD in rats. a CSD was induced by topical application of 2 M KCl for 30 min onto cerebral cortex with dura intact via the posterior burr hole on the right parietal bone. The ipsilateral anterior hole was used for CSD recording. The anti-TRPA1 antibody (i, n = 10) or anti-IgG antibody (ii, n = 8) was perfused through a cannula implanted in the contralateral ventricle (i.c.v) at 4 days prior to CSD induction. In the sham group (iii), the anti-IgG antibody was i.c.v perfused in the absence of KCl application as the control (n = 7). The whole ipsilateral cerebral cortical tissue was subsequently used for detecting MDA level immediately after the in vivo experiment. b A representative trace showing CSD propagation wave after i.c.v perfusion of the anti-IgG antibody. CSD number, latency (minute) and magnitude (area under the curve of each CSD wave, mV × minute) were used for quantifying the excitation phase of CSD. The effects of the anti-TRPA1 antibody at 0.8 μg on CSD number are shown in panel (c), latency in panel (d) and magnitude in panel (e). All the values shown are median (range). *p < 0.05, Mann-Whitney U test with one-tailed calculation was used for comparison of the anti-IgG antibody and the anti-TRPA1 antibody group
Fig. 4
Fig. 4
Effects of the anti-TRPA1 antibody on MDA level (μmol/mg protein) induced by CSD in the ipsilateral cerebral cortex of rat and correlation analysis of each CSD characteristic with levels of cortical ipsilateral MDA between the anti-TRPA1 antibody or anti-IgG antibody groups. a CSD promoted ipsilateral cortical MDA level, which was inhibited by the pretreatment of anti-TRPA1 antibody perfused into the contralateral i.c.v in rats. Mann-Whitney U test, one-tailed, for significance between each group (*p < 0.05, ***p < 0.001). The reduced CSD magnitude (d), but not CSD number (b) and latency (c) positively correlated with a lower MDA level after the anti-TRPA1 antibody perfusion. Red dotted lines indicated positive correlation between CSD magnitude and MDA level
Fig. 5
Fig. 5
Effects of modulation of ROS and TRPA1 on cortical susceptibility to CSD and their interaction during CSD in the mouse brain slice. Submaximal CSD was induced by KCl at 50 mM. There were six groups: Kreb’s (i, n = 6) as control, 3 mM of the ROS inhibitor, tempol (ii, n = 7), 50 μM of ROS activator, H2O2 (iii, n = 6), 3 mM of tempol with 50 μM of H2O2 (vi, n = 8) or with 15 μM of the TRPA1 agonist, UMB (v, n = 7), 50 μM of H2O2 with 1 μM of the TRPA1 antagonist, A967079 (vi, n = 6). In order to minimize the animal use, data in Kreb’s control group were adopted and transformed from that in Fig. 4 in our previous paper [5]. Representative trace of the 2nd CSD episode in each group were shown in the panel a. The data showed that cortical susceptibility to CSD is suppressed by tempol, facilitated by H2O2, and there is a bidirectional interaction between ROS and TRPA1 in modulating latency (b) and magnitude (c) of CSD. CSD latency (second) was given as median (rang). CSD magnitude were plotted as percentage of their initial levels (1st CSD episode) and indicated as median (range). Mann-Whitney U test (one-tailed) was for significant analysis between two independent groups. * p < 0.05, ** p < 0.01, ***p < 0.001 indicate significance
Fig. 6
Fig. 6
Both ROS and the TRPA1 activation reversed the inhibitory effects of the anti-CGRP antibody on CSD in the mouse brain slice. CSD was induced by 260 mM KCl. There were four groups: anti-IgG antibody at 0.025 μM (i, n = 6) as the control, anti-CGRP antibody at 0.4 μM in the absence (ii, n = 6) or presence of 50 μM of the TRPA1 agonist, AITC (iii, n = 6) or the ROS activator, H2O2 (vi, n = 6). In order to minimize the animal use, data in anti-IgG antibody control group and the anti-CGRP antibody were adopted and transformed from that in Fig. 5 in our recent paper [5]. Representative trace of the 2nd CSD episode in each group are shown in the panel a. The data showed that both ROS and the TRPA1 activation reversed the prolonged CSD latency (b), but not magnitude (c) under the perfusion of the anti-CGRP antibody. Data were plotted as percentage of their initial levels (1st CSD episode) and indicated as median (range). Mann-Whitney U test, one-tailed, was used for significant analysis between two independent groups. *p < 0.05, **p < 0.01. Abbreviation: Ab indicates antibody
Fig. 7
Fig. 7
A diagram depicting ROS/TRPA1/CGRP signaling in modulating cortical susceptibility to CSD. It is proposed that ROS may trigger TRPA1 activation and CGRP production, which create a positive feedback loop in regulating cortical susceptibility to CSD. In which way, ROS could facilitate CSD propagation for the subsequent development of migraine

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