NOP receptor agonist attenuates nitroglycerin-induced migraine-like symptoms in mice

Abstract

Migraine is an extraordinarily prevalent and disabling headache disorder that affects one billion people worldwide. Throbbing pain is one of several migraine symptoms including sensitivity to light (photophobia), sometimes to sounds, smell and touch. The basic mechanisms underlying migraine remain inadequately understood, and current treatments (with triptans being the primary standard of care) are not well tolerated by some patients. NOP (Nociceptin OPioid) receptors, the fourth member of the opioid receptor family, are expressed in the brain and periphery with particularly high expression known to be in trigeminal ganglia (TG). The aim of our study was to further explore the involvement of the NOP receptor system in migraine. To this end, we used immunohistochemistry to examine NOP receptor distribution in TG and trigeminal nucleus caudalus (TNC) in mice, including colocalization with specific cellular markers, and used nitroglycerin (NTG) models of migraine to assess the influence of the selective NOP receptor agonist, Ro 64-6198, on NTG-induced pain (sensitivity of paw and head using von Frey filaments) and photophobia in mice. Our immunohistochemical studies with NOP-eGFP knock-in mice indicate that NOP receptors are on the majority of neurons in the TG and are also very highly expressed in the TNC. In addition, Ro 64-6198 can dose dependently block NTG-induced paw and head allodynia, an effect that is blocked by the NOP antagonist, SB-612111. Moreover, Ro 64-6198, can decrease NTG-induced light sensitivity in mice. These results suggest that NOP receptor agonists should be futher explored as treatment for migraine symptoms. This article is part of the special issue on Neuropeptides.

Keywords: Cephalic pain; Glyceryl trinitrate; NOP receptor; Nitroglycerine; Ro 64-6198; Trigeminal ganglia.

Figure 1.. NOP-eGFP receptor expression in the trigeminal nucleus caudalis.

Representative images show NOP-eGFP expression in the TNC (Sp5C, spinal trigeminal nucleus caudalis). CGRP was used as a maker to visualize spinal trigeminal tract (Sp5). Scale bar, 250 μm.

Figure 2.. Neuroanatomical analysis of TG neurons using NOP-eGFP mice.

To characterize the trigeminal neurons expressing NOP-eGFP, sections were incubated with anti-GFP antibody together with cellular markers. A, The NOP-eGFP containing trigeminal neurons were quantified by determining the percentage of eGFP-positive cells (green) compared with the total number of trigeminal sensory neurons (n = 5264 neurons). Nuclei were stained with DAPI (blue). The total number of trigeminal sensory neurons was determined by counting the total number of DAPI-stained cells and excluding those from satellite cells. Tissue sections were also co-stained with anti-CGRP and -NF200 antibodies (B), anti-nNOS and -CGRP (C), biotinylated IB4 together with streptavidin conjugated with Alexa fluor (D), anti-Ret and -NF200 (E) and anti-TrkC and -NF200 (F). Yellow arrowheads indicate the trigeminal neurons where co-staining occurs. Scale bars, 100 μm.

Figure 3.. The NOP receptor agonist Ro 64-6198 attenuates NTG-induced mechanical allodynia, an effect blocked by the NOP antagonist SB-612111.

Effect of Ro 64-6198 (0.3 to 3.0 mg/kg) on NTG-induced mechanical hypersensitivity in (A) the periorbital region and (B) the paw. (C) SB-612111 (10 mg/kg) effect on Ro 64-6198 in mice with NTG-induced pain. NTG-induced hypersensitivity in the head and the paw was measured by von Frey filaments. (A) NTG (10 mg/kg) produced marked mechanical response (***p<0.001). Ro 64-6198 was effective in blocking NTG-induced allodynia at both 1.0 and 3.0 mg/kg doses (###p<0.001 for both, n=6–12 mice per dose). (B) NTG (10 mg/kg) also produced marked mechanical response in the paw (**p<0.01), with Ro 64-6198 being effective in reversing allodynia at 1.0 mg/kg dose (### p<0.001). (C) In a separate experiment, Ro 64-6198 blocked mechanical allodynia induced by NTG (^^ p<0.01). This effect was reversed by the antagonist SB-612111 (## p<0.01, two-way ANOVA followed by Newman-Keuls’s post hoc test). Experiments shown in B and C were carried out using n=10–12 mice (4–6 males and 5–6 females) per group and n=8 per group, respectively.

Figure 4.. NTG-induced light aversion is blocked by Ro 64-6198

Effects of Ro 64-6198 and sumatriptan on NTG-induced light sensitivity in the light/dark box in male and female mice. (A) NTG-treated animals showed decrease in the percent (%) of time spent in light chamber (p < 0.05, n = 7 per group). In a separate experiment, Ro 64-6198 (0.3 mg/kg, i.p.) blocked NTG-induced light aversion (*p <0.05) in (B) male and (C) female mice (n = 8–10 per group, ## p < 0.01 and # p < 0.05, respectively, when compared to NTG-treated mice; two-way ANOVA followed by Newman-Keuls’s post hoc test). Sumatriptan (0.6 mg/kg, i.p.) did not reverse NTG-induced light aversion in male (D) and female (E) mice (main NTG effect: +p<0.05 for both, n = 7 per group).

Figure 5.. Treatment of NTG does not increase anxiety-like behavior.

Anxiety-like behavior was measured by the elevated plus maze test in male (A and B) and female mice (C and D). The percent (%) of time spent in the open arms and % of open-arm entries was calculated (mean ± standard error of the mean (SEM)) for each group. (A) % of time spent in the open arms was increased by Ro 64-6198 0.3 mg/kg (main Ro 64-6198 effect: ⁺ p < 0.05) and decreased by NTG (10 mg/kg) in male mice (main NTG effect: ⁺⁺ p < 0.01). (B) % open arm entries was decreased by NTG (10 mg/kg) in male mice (main NTG effect: ⁺ p < 0.05, n = 7 per group). (C, D) Anxiety-like response was not altered by Ro 64-6198 (0.3 mg/kg) in female mice (n = 8–9 per group).