Role of phosphorylated extracellular signal-regulated kinase, calcitonin gene-related peptide and cyclooxygenase-2 in experimental rat models of migraine

Abstract

Although migraine is a common neurological condition, the pathomechanism is not yet fully understood. Activation of the trigeminovascular system (TVS) has an important function in this disorder and neurogenic inflammation and central sensitization are important mechanisms underlying this condition. Nitroglycerin (NTG) infusion in rats closely mimics a universally accepted human model of migraine. Electrical stimulation of the trigeminal ganglion (ESTG) of rats can also activate TVS during a migraine attack. Numerous studies have revealed that phosphorylated extracellular signal-regulated kinase (p-ERK), calcitonin gene-related peptide (CGRP) and cyclooxygenase-2 (COX-2) are involved in pain and nociceptive pathways. However, few studies have examined whether p-ERK, CGRP and COX-2 are involved in neurogenic inflammation and central sensitization. In the present study, the expression of p-ERK, CGRP and COX-2 was detected in the dura mater, trigeminal ganglion (TG) and spinal trigeminal nucleus caudalis in NTG-induced rats and ESTG models by immunohistochemistry. The three areas considered were crucial components of the TVS. The selective COX-2 inhibitor nimesulide was used in ESTG rats to examine the association between p-ERK, CGRP and COX-2. The results demonstrated that p‑ERK, CGRP and COX-2 mediated neurogenic inflammation and central sensitization in migraine. In addition, the expression of p-ERK and CGRP was attenuated by the COX-2 inhibitor.

Figure 1

Representative images of p-ERK, CGRP and COX-2 immunoreactivity in the dura mater 30 min after (A–C) NTG or (D–F) vehicle infusion by immunohistochemistry and (G) analysis of the MOD of p-ERK, CGRP and COX-2 expression in the dura mater. An increase in (A) p-ERK, (B) CGRP and (C) COX-2 expression was observed in the dura mater following NTG infusion and the MOD value for NTG-treated rats was significantly higher than in the vehicle and blank groups. (^###P<0.001, compared with the vehicle and blank groups; n=6 in each group; error bars indicate standard deviation; scale bar=100 µm). MOD, mean optical density; p-ERK, phosphorylated extracellular signal-regulated kinase; CGRP, calcitonin gene-related peptide; COX-2, cyclooxygenase-2; NTG, nitroglycerin.

Figure 2

Representative images of p-ERK, CGRP and COX-2 immunoreactivity in the TG 30 min after (A–C) NTG or (D–F) vehicle infusion by immunohistochemistry and (G) analysis of the MOD of p-ERK, CGRP and COX-2 expression in the TG. An increase in (A) p-ERK, (B) CGRP and (C) COX-2 expression was observed in the TG following NTG infusion and the MOD value for NTG-treated rats was significantly higher than in the vehicle and blank groups. (^###P<0.001, compared with the vehicle and blank groups; n=6 in each group; error bars indicate standard deviation; scale bar=100 µm). MOD, mean optical density; p-ERK, phosphorylated extracellular signal-regulated kinase; CGRP, calcitonin gene-related peptide; COX-2, cyclooxygenase-2; TG, trigeminal ganglion; NTG, nitroglycerin.

Figure 3

Representative images of p-ERK, CGRP and COX-2 immunoreactivity in the TNC 30 min after (A–C) NTG or (D–F) vehicle infusion by immunohistochemistry, (G) rat TNC section nuclei and (H) analysis of the MOD of p-ERK, CGRP and COX-2 expression in the TG. An increase in (A) p-ERK, (B) CGRP and (C) COX-2 expression was observed in the TNC following NTG infusion and the MOD value for NTG-treated rats was significantly higher than in the vehicle and blank groups. [^###P<0.001, compared with the vehicle and blank groups; n=6 in each group; error bars indicate standard deviation; scale bar (A–F)=100 µm; scale bar (G)=1 mm]. MOD, mean optical density; p-ERK, phosphorylated extracellular signal-regulated kinase; CGRP, calcitonin gene-related peptide; COX-2, cyclooxygenase-2; TNC, trigeminal nucleus caudalis; NTG, nitroglycerin.

Figure 4

Expression of p-ERK in the (A) dura mater, (B) TG and (C) TNC following NTG infusion. As shown in the histogram, the phosphorylation of ERK following NTG infusion of the dura mater, TG and TNC demonstrated a temporal profile. Significantly higher levels of p-ERK were found in the dura mater, TG and TNC 30 min after NTG administration compared with the controls. The p-ERK levels gradually decreased and were close to the basal level by 3 h. (^**P<0.01, compared with the NS group; n=6 in each group; error bars indicate standard deviation). p-ERK, phosphorylated extracellular signal-regulated kinase; TNC, trigeminal nucleus caudalis; TG, trigeminal ganglion; NTG, nitroglycerin; NS, vehicle-treated rats.

Figure 5

Effect of electrical stimulation and pretreatment with NM on the protein expression of p-ERK, CGRP and COX-2 in the (A) dura mater, (B) TG and (C) TNC. As shown in the histogram, following electrical stimulation, a significant increase in p-ERK, CGRP and COX-2 was observed in the TNC, ipsilateral dura mater and TG compared with the sham-surgery and blank groups. Pretreatment with NM demonstrated a significant decrease in p-ERK, CGRP and COX-2 MOD values in the TNC, ipsilateral dura mater and TG compared with the electrically-stimulated rats, however this was higher than in the sham-surgery and blank groups. No differences were detected between the sham-surgery and the blank groups. (^*P<0.05, compared with the electrically-stimulated group; ^###P<0.001, compared with the sham-surgery and blank groups; n=6 in each group; error bars indicate standard deviation). MOD, mean optical density; p-ERK, phosphorylated extracellular signal-regulated kinase; CGRP, calcitonin gene-related peptide; COX-2, cyclooxygenase-2; NM, nimesulide; ESTG, electrical stimulation of the trigeminal ganglion; SO, sham operation; TNC, trigeminal nucleus caudalis; TG, trigeminal ganglion.

Figure 6

Connections between p-ERK, CGRP and COX-2 in the pathophysi-ological mechanisms of migraine. Electrical stimulation of the TG leads to neurogenic inflammation in trigeminal neurons and glial cells followed by mast cell degranulation. Mast cell degranulation in turn activates mast cells to release COX-2 followed by synthesis of PGE₂. Newly synthesized PGE₂ induces trigeminal neurons to release CGRP and also induces TRPV1 sensitization. The influx of Ca²⁺ via TRPV1 upregulates the level of p-ERK in the TG and causes peripheral and central hypersensitivity, which induces migraine attack and pain. p-ERK, phosphorylated extracellular signal-regulated kinase; CGRP, calcitonin gene-related peptide; COX-2, cyclooxygenase-2; TRPV1, transient receptor potential vanilloid receptor 1; TG, trigeminal ganglion; PGE₂, prostaglandin E₂.