Effect of dural inflammatory soup application on activation and sensitization markers in the caudal trigeminal nucleus of the rat and the modulatory effects of sumatriptan and kynurenic acid

Abstract

Background: The topical inflammatory soup can model the inflammation of the dura mater causing hypersensitivity and activation of the trigeminal system, a phenomenon present in migraineurs. Calcitonin gene-related peptide, transient receptor potential vanilloid-1 receptor, and neuronal nitric oxide synthase are important in the sensitization process there. 5-HT_1B/1D receptor agonists, triptans are used as a treatment of migraine. Kynurenic acid an NMDA antagonist can act on structures involved in trigeminal activation.

Aim: We investigated the effect of inflammatory soup induced dural inflammation on the calcitonin gene-related peptide, transient receptor potential vanilloid-1 receptor, and neuronal nitric oxide synthase levels in the caudal trigeminal nucleus. We also tested whether pretreatment with a well-known antimigraine drug, such as sumatriptan and kynurenic acid, a compound with a different mechanism of action, can affect these changes and if their modulatory effects are comparable.

Material and methods: After subcutaneous sumatriptan or intraperitoneal kynurenic acid the dura mater of adult male Sprague-Dawley rats (n = 72) was treated with inflammatory soup or its vehicle (synthetic interstitial fluid). Two and a half or four hours later perfusion was performed and the caudal trigeminal nucleus was removed for immunohistochemistry.

Results and conclusion: Inflammatory soup increased calcitonin gene-related peptide, transient receptor potential vanilloid-1 receptor, and neuronal nitric oxide synthase in the caudal trigeminal nucleus compared to placebo, which was attenuated by sumatriptan and kynurenic acid. This suggests the involvement of 5-HT_1B/1D and NMDA receptors in neurogenic inflammation development of the dura and thus in migraine attacks.

Keywords: CGRP; Inflammatory soup; Kynurenic acid; Migraine; Sumatriptan; TRPV1; Trigeminal system; nNOS.

Fig. 1

CGRP immunoreactivity 2.5 h after IS treatment Representative photomicrographs of the CGRP expression in the trigemino-cervical segments after 2.5 h. a – SIF, b – IS, c – SUMASIF, d – SUMAIS, e – KYNSIF, f – KYNIS. In the IS group, the CGRP staining was stronger than in the placebo group. Sumatriptan and kynurenic acid were able to attenuate this effect. SIF: synthetic interstitial fluid, IS: inflammatory soup, SUMASIF: sumatriptan + synthetic interstitial fluid, SUMAIS: sumatriptan + inflammatory soup, KYNSIF: kynurenic acid + synthetic interstitial fluid, KYNIS: kynurenic acid + inflammatory soup. Scale bars: 200 μm, 50 μm

Fig. 2

CGRP immunoreactivity 4 h after IS treatment Representative photomicrographs of the CGRP expression in the trigemino-cervical segments after 4 h. a – SIF, b – IS, c – SUMASIF, d – SUMAIS, e – KYNSIF, f – KYNIS. In the IS group, the CGRP staining was stronger than in the placebo group. Sumatriptan and kynurenic acid were able to attenuate this effect. SIF: synthetic interstitial fluid, IS: inflammatory soup, SUMASIF: sumatriptan + synthetic interstitial fluid, SUMAIS: sumatriptan + inflammatory soup, KYNSIF: kynurenic acid + synthetic interstitial fluid, KYNIS: kynurenic acid + inflammatory soup. Scale bars: 200 μm, 50 μm

Fig. 3

Statistical analysis of CGRP staining 2.5 and 4 h after IS treatment The quantitative analysis shows that in the IS group the area covered by fibers showing CGRP positivity is significantly higher than in the control group in both timepoints. a 2.5 h after IS treatment, sumatriptan was able to attenuate this effect in the V/1 area. b Similar to sumatriptan kynurenic acid weakened the effect of IS in the V/1 area. c 4 h after IS treatment, sumatriptan was able to mitigate this effect in the and V/1 area. d Kynurenic acid decreased the effect of IS in the V/1 area. *p < 0.05; **p < 0.01, ***p < 0.001 * means SIF-IS differences, + means IS-SUMA/KYNA. SIF: synthetic interstitial fluid, IS: inflammatory soup, SUMASIF: sumatriptan + synthetic interstitial fluid, SUMAIS: sumatriptan + inflammatory soup, KYNSIF: kynurenic acid + synthetic interstitial fluid, KYNIS: kynurenic acid + inflammatory soup

Fig. 4

TRPV1 immunoreactivity 4 h after IS treatment Representative photomicrographs of the TRPV1 expression in the trigemino-cervical segments after 4 h. a – SIF, b – IS, c – SUMASIF, d – SUMAIS, e – KYNSIF, f – KYNIS. In the IS group, the area covered by TRPV1 was higher than in the placebo group. Sumatriptan and kynurenic acid were able to attenuate this effect. SIF: synthetic interstitial fluid, IS: inflammatory soup, SUMASIF: sumatriptan + synthetic interstitial fluid, SUMAIS: sumatriptan + inflammatory soup, KYNSIF: kynurenic acid + synthetic interstitial fluid, KYNIS: kynurenic acid + inflammatory soup. Scale bars: 200 μm, 50 μm

Fig. 5

nNOS immunoreactivity 4 h after IS treatment Representative photomicrographs of the nNOS expression in the trigemino-cervical segments after 4 h. a – SIF, b – IS, c – SUMASIF, d – SUMAIS, e – KYNSIF, f – KYNIS. In the IS group, the number of nNOS-IR cells was increased compared to the SIF- treated group. Sumatriptan and kynurenic acid were able to mitigate this effect. SIF: synthetic interstitial fluid, IS: inflammatory soup, SUMASIF: sumatriptan + synthetic interstitial fluid, SUMAIS: sumatriptan + inflammatory soup, KYNSIF: kynurenic acid + synthetic interstitial fluid, KYNIS: kynurenic acid + inflammatory soup. Scale bars: 200 μm, 50 μm.

Fig. 6

Statistical analysis of TRPV1 and nNOS staining 4 h after IS treatment The quantitative analysis shows that in the IS group the area covered by TRPV1 IR fibers and the number of nNOS IR cells is significantly higher than in the control group after 4 h. a Sumatriptan was able to attenuate the increase in TRPV1 IR fibers in the V/1 area. b Similar to sumatriptan kynurenic acid also mitigated the effect of IS in the V/1 area. c In the V/1 area sumatriptan was able to abolish the increase in nNOS IR cells. d Kynurenic acid also weakened the effect of IS in the V/1 area *p < 0.05; **p < 0.01, ***p < 0.001. * means SIF-IS differences, + means IS-SUMA/KYNA differences. SIF: synthetic interstitial fluid, IS: inflammatory soup, SUMASIF: sumatriptan + synthetic interstitial fluid, SUMAIS: sumatriptan + inflammatory soup, KYNSIF: kynurenic acid + synthetic interstitial fluid, KYNIS: kynurenic acid + inflammatory soup