Up-regulation of calcitonin gene-related peptide in trigeminal ganglion following chronic exposure to paracetamol in a CSD migraine animal model

Highlights

• Short-term treatment with APAP does not have any effect on the CGRP expression in TG.

• Short-term treatment with APAP can attenuate the CGRP expression in TG induced by CSD activation.

• Long-term treatment with APAP can up-regulate CGRP expression in TG.

• Up-regulation of CGRP is enhanced in long term APAP treatment in combination with CSD activation.

Abstract

Previously, our group has demonstrated that chronic paracetamol (APAP) treatment induces alterations to the trigeminovascular nociceptive system in the cortical spreading depression (CSD) migraine animal model. The calcitonin gene related peptide (CGRP) is a key neuropeptide involved in the activation of the trigeminovascular nociceptive system. Therefore, this study examined the expression levels of CGRP in the trigeminal ganglion (TG) after chronic APAP exposure (0, 15, and 30 days) using a CSD model. Rats were divided into control, CSD only, APAP only and APAP treatment with CSD groups. A single injection (i.p.) of APAP (200 mg/kg body weight) was given to the 0-day APAP-treated groups, while the other APAP-treated groups received daily injections for 15 and 30 days. CSD was induced by the topical application of KCl to the parietal cortex. The protein expression of CGRP in the TG was evaluated by immunohistochemistry, and the CGRP mRNA level was investigated by real-time quantitative reverse transcription polymerase chain reaction. The results revealed that the induction of CSD significantly increased the level of CGRP protein but had no effect on CGRP mRNA level. Pretreatment with APAP 1 hour before CSD activation significantly reduced CGRP expression induced by CSD. In contrast, chronic treatment with APAP (15 and 30 days) significantly enhanced CGRP expression in both protein and mRNA levels when compared with the control groups. In combination with CSD, the expression of CGRP further increased in the animal with 30 day treatment. These findings indicate that chronic treatment with APAP induces an increase of CGRP expression in the TG. This alteration may be associated with the increased trigeminovascular nociception observed in our previous studies.

Introduction

Paracetamol (acetaminophen, APAP) is one of the most commonly used medicines for the treatment of pain-associated symptoms. Several lines of evidence have demonstrated that this drug efficiently controls a variety of chronic pain, including migraine headaches.

Although the prominent analgesic effect of APAP is expected to act in the central nervous system (CNS), the exact mechanism of action is still under debate. Several mechanisms have been proposed to underlie the analgesic effects of APAP, including inhibition of prostaglandin synthesis, promotion of the serotonergic descending inhibitory pathways, association with endocannabinoid signaling, and interaction with the dynorphin and opioid systems (Pickering et al, 2006, Sandrini et al, 2007, Smith, 2009). Regarding the effect of this drug treatment, several studies have demonstrated that the treatment with APAP within the therapeutic dosage for short-term, most of them, demonstrated the protective effect against several pathological stimuli including oxidative stress (Tripathy, Grammas, 2009a, Tripathy, Grammas, 2009b) and aging (Tripathy et al., 2012). The anti-inflammation and anti-oxidative stress properties of this drug have been explained as mechanisms underlying these protective effects (Tripathy, Grammas, 2009a, Tripathy, Grammas, 2009b).

However, several researches have demonstrated the adverse effect of this drug when used as a long-term treatment. The study by Fakunle et al. in hippocampus had demonstrated that the rat with chronic APAP treatment (100 mg/kg body weight for 6 weeks) in combination with alcohol intake resulted in the degeneration of hippocampal neurons which associated with the deficit of learning and memory (Fakunle et al., 2011). In addition, the cohort study in Norwegian mother and child recently revealed that long-term exposure with APAP (more than 28 days) in the prenatal period could induce the deficit of gross motor functioning and communication action skills in children (Brandlistuen et al., 2013).

Moreover, the results from our previous study in the cortical spreading depression (CSD) model were in line with those observations. CSD is well accepted as one of standard animal model for studying migraine pathophysiology which involves the activation of the trigeminovascular nociceptive system (Eikermann-Haerter, Moskowitz, 2008, Jander et al, 2001, Shatillo et al, 2013). In our previous studies, the trigeminovascular nociceptive system responded differently between acute and chronic treatment with APAP in both neural and vascular compartments. While short-term treatment with APAP (200 mg/kg body weight) could attenuate the CSD-induced alteration in the trigeminal nociception (Supornsilpchai et al, 2010a, Supornsilpchai et al, 2010b) and cerebral microvessels (Yisarakun et al., 2014), the opposite responses were observed in the rat with chronic treatment. The greater degree of the alteration in both cortical neurons (Supornsilpchai et al, 2010a, Supornsilpchai et al, 2010b) and cerebral microvessels (Yisarakun et al., 2014) induced by CSD were observed in those with chronic APAP treatment (Supornsilpchai et al, 2010a, Supornsilpchai et al, 2010b, Yisarakun et al, 2014). Based on these results, we have hypothesized that the treatment with APAP for long period may alter the trigeminovascular nociceptive control.

Calcitonin gene-related peptide (CGRP) is a neuropeptide consisting of 37 amino acids with two isoforms, α- and β-CGRP. While α-CGRP is widely distributed in both peripheral and central nervous systems (Rosenfeld et al., 1983), β-CGRP is found in the enteric nervous system and the pituitary gland (Sternini, 1992). In the trigeminal ganglion (TG), a cluster of the cell bodies of trigeminal neurons, α-CGRP is predominantly expressed (Amara et al, 1985, Durham, Vause, 2010). CGRP is a key neuropeptide involved in both neural and vascular alterations and its involvement in migraine pathophysiology is well accepted (Arulmani et al, 2004, Durham, 2008, Durham, Vause, 2010). The role of this neuropeptide in trigeminovascular nociception has been confirmed by the discovery of an association between the sensitization of nociceptive systems (both central and peripheral) and the release of CGRP from the TG (Cady et al, 2011, De Felice, Porreca, 2009, De Felice et al, 2010). Based on these cumulative data, a robust interest in CGRP alterations in several pathologic conditions with abnormal nociceptive responses has been demonstrated (Belanger et al, 2002, Bowen et al, 2006, Eberhardt et al, 2014, Ma et al, 2001, Wang et al, 2010).

In order to determine whether the hypersensitivity of trigeminovascular nociception observed in our previous studies (Supornsilpchai et al, 2010a, Supornsilpchai et al, 2010b) resulted from alterations in the expression of CGRP in the TG, we examined the effects of chronic APAP treatment at three different time points (0, 15, and 30 days) in a CSD migraine animal model. The expression levels of CGRP protein and mRNA in all experimental groups were investigated by immunohistochemistry and real-time quantitative reverse transcription polymerase chain reaction, respectively.